CHEMICAL METHODS

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CHEMICAL METHODS
OF DETECTING
MICROORGANISMS
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Limulus Amoebocyte Lysate (LAL) assay
Adenosine-Triphophate (ATP) assay
Fluoro- and chromogenic substrate
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Limulus Amoebocyte Lysate
(LAL) assay

• is able to detect only compounds of gram negative
  bacteria (endotoxins).

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"endotoxin" refer to the lipopolysaccharide (LPS)
complex associated with the outer membrane of
Gram-negative bacteria.
Endotoxins are part of the outer membrane of the
cell wall of Gram-negative bacteria whether the
organisms are pathogens or not.
Endotoxins can cause symptoms such as fever and
ache
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Salmonella
Neisseria
E. coli
Shigella
Pseudomonas
Haemophilus
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What is Limulus Amoebocyte Lysate

• Limulus Amebocyte Lysate (LAL), derived
• from circulating blood cells (amebocytes) of
• the horseshoe crab.
• An enzyme isolated from the crab's blood called
  limulus amoebocyte lysate (LAL)

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• The blood is a mixture of liquid serum and
  suspended amoebocytes.
• The crab's blood is centrifuged to separate the
  amoebocytes from the liquid plasma.
• The conditions for extracting blood must be
  sterile.

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• It is the presence of the blood clot that signals
  that an endotoxin is present.

• A suspect sample is mixed with reconstituted LAL
  and allowed to sit in a small tube.

45 min.

LAL
45 min.

• A Limulus amoebocyte lysate (LAL) assay can take
  as little as 45 minutes.

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• for use in the testing of drugs, blood products,
  intravenous fluids, and disposable pharmaceutical
  devices.
• Its uses include diagnosis of endotoxemia in
  conjunction with cirrhosis, cancer, meningitis,
  eye disease, dental problems, gonorrhea,
  boutonneuse fever, water quality analysis,
  urinary tract infections, and bacterially
  contaminated meat, fish, and dairy products.

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Adenosine-Triphophate
(ATP) assay

• Indicate Total Viable Biomass
• ATP is a chemical found in all living cells. 
• All living organisms contain and use ATP as their
  main energy source.
• Larger cells show higher ATP values and this
  affects the final microbial counts 
• When a cell dies, ATP production is stopped and
  the ATP which has been made is rapidly degraded. 
  
• When there is no ATP detected, it is generally
  accepted that there are no more living cells.

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Adenosine Triphosphate
(ATP) assay

• This kit contains synthetic D-luciferin, the
  primary substrate of the firefly luciferase light
  producing system.

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• The procedure begins by applying a detergent to
  the sample, to break open whatever cells it may
  contain and release their ATP. Then chemicals are
  added that emit light in the presence of ATP,
  signaling that the sample contained one or more
  living organisms.

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Luciferase
Luciferin
Luciferin luciferase AMP complex
O2
ATP
PPi
CO2
Oxidized luciferin-luciferase-AMP complex
(activated)
Light
Oxidized luciferin-luciferase-AMP complex
(unexcited ground state)
During the reaction, ATP is transformed into
adenosine monophosphate (AMP) and light. The
intensity of the light produced is directly
proportional to the cell concentration of ATP.
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Adenosine Triphosphate (ATP) assay (cont.)

• This light is measured by instruments called
  luminometers, which essentially consist of a
  photodetector, a signal amplifier, and a signal
  processor.

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Fluoro - and Chromogenic
substrates

• Detection of activities of specific enzymes.
• Detection, enumeration, and idenification of
  viable bacteria from a sample.
• The substrate is hydrolyzed by the specific
  enzyme, yielding free fluoro- and chromopores.

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Simple detection by blue-green color fluorecence
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Fluoro- and Chromogenic
Substrates

• 4-metylbelliferyl-ß-D-glucuronide (MUG)
• 4-methylumbelliferyl-ß-D-galactoside (MUGal)
• 4-bromo-4-chloro-3-indoyl-ß-D-glucuronide
  (X-GLUC)
• O-nitrophenyl-ß-D-galactopyranoside (ONPG)
• 5-bromo-4-chloro-3-indoxyl-ß-D-gluconide (BCIG)
• L-alanine-p-nitroaniline (LAPN)

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4-metylbelliferyl-ß-D-glucuronide (MUG)

• M U G is hydrolyzed by ß-D-glucuronidase (G U D)
  to release the fluorescent 4-metylbelliferyl
  moiety which is deteced wih long-wave ultraviolet
  light
• E. coli detection
• E. coli is indicated by M U G fluorescence.
• A few salmonellae and shigellae are G U D
  positive, as are some corynebacteria.

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4-methylumbelliferyl-ß-D-galactoside (MUGal)

• Detection of fecal coliforms in water.
• Also used to differentiate species of
  enterococci.
• By observing for strach hydrolysis and
  fluorescence, 86 of enterococci from
  enviromental samples were correctly
  differentiated.

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4-bromo-4-chloro-3-indoyl-ß-D-glucuronide
(X-GLUC)

• Detection of E. coli
• With this substrste added at 500 ppm to a
  peptone-Tergitol agar, E. coli produced a blue
  color in 24 hours that did not diffuse from
  colonies and did not require fluorescent light.
• When compared with a standard M P N on 50 ground
  beef sample tube, no differences were observed
  between the two methods.

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O-nitrophenyl-ß-D-galactopyranoside (ONPG)

• To deter mine E. coli in water
• Tubes that contain colifor ms become yellow.

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5-bromo-4-chloro-3-indoxyl-ß-D-gluconide (BCIG)

• Results in E. coli colonies being blue, while
  other organisms are not blue.

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L-alanine-p-nitroaniline (LAPN)

• Specific for gram-negative bacteria on the
  premise that aminopeptidase is restricted to this
  group.
• Te enzyme cleaves L-nitroanilide to yield
  p-nitroaniline, a yellow compound that is read
  specrophotometrically at 390 nm.

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THANK YOU
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Ms. Kanyapak Sapsanyakorn
I.D. 4518517