MCB 401 Analytical Microbiology and Quality control

Microbial Toxicity test/assay

Outline

● The use of microbial responses to assess chemical toxicity against

microorganisms

i. Microbial growth

ii.Enzyme activity and biosynthesis

iii.Bioluminescence

● Determination of toxicity thresholds, LC50

● Detection of pollutants using bacterial bioluminescence, e.g Heavy metals, PAHs

etc

Advantages

i.Low cost ii.Rapid response to toxicants iii.High sample throughput iv.Modest

laboratory equipment and space requirement v.Low sample volume vi.Portability
and reproducible response

Attributes of good responses

i. The capability of accurate measurement. ii. Small variation of sensitivity within

population iii.Rapid development of response iv. Show of gradation with graded
concentration of active substance

What are some microbial responses to toxicity?

•Cell viability-Microbial growth
•Metabolite production-enzyme activities, microbial activities
•Light emission-bioluminescence
MICROBIAL GROWTH

Direct and indirect determination of microbial growth

-Viable cell count (serial dilution, heterotrophic count or colony count)

-Turbidity measurement (spectrophotometer or calorimeter)

• Absorbance or optical density is obtained

•Plot against viable cell count to obtain a correlation equation for future
estimations

Turbidity Measurement Using Spectrometer

As light passes through a material, it is absorbed by molecules in the material.

Turbidity can be determined using Colorimeter or Spectrophotometer by
measuring the decrease in transmitted light due to blockage by particles (ie toxins,
impurities). In turbidity measurement using spectrophotometer, you obtain
Absorbance (A) or Optical Density (OD). Anything that makes water cloudy will
increase turbidity. High turbidity can be caused by chemicals in the water, mud,
silt etc. turbidity affects the growth rate because increased turbidity causes a
decrease in the amount of light for photosynthesis as found in Algae.

Transmittance (T): is the fraction of incident light which is transmitted. That is,
the amount of light that successfully passes through the substance and comes out
the other side.

Absorbance (A): states how much of the light the sample absorbed. That is,
amount of light absorbed. When much light is passing through the sample, little
will be absorbed i.e Low % T = High A. If all the light passes through a solution
without any absorption, then Absorbance (A)= Zero, and T = is 100%. If all the
light is absorbed, then % T = Zero and A is infinite.

Calculation of Absorbance:
1 Io I I Io
T α A, A= I , T =I o, A = Log10 T , A = Log10 I , A = -Log10 T
A= Absorbance, I0 = Initial light intensity before transmission via sample,
I=Intensity of light after transmission via sample, T= Transmittance (%).

Direct And Indirect Cell Count Determination

Use Of Nucleic Acids:

•qPCR, DAPI staining, fluorescent in situ hybridization (FISH)

•DAPI staining and FISH-Nucleic acids are labelled with fluorescing dyes such as
DAPI and Acridine orange and viewed using epifluorescence microscope

•DAPI binds to double stranded DNA and capture all cells irrespective of their
metabolic state

•FISH targets ribosomal RNA and hence only active microoganismsare captured
•The use of nucleic acid captures the non-culturable cells

•qPCR involves the quantification of DNA samples extracted from a sample

•qPCR does not rely of visualization, separation of cells and viability of cell.
Use of metabolites:
•CO2 emission (respiration)
•Acid production-pH change, High Performance Liquid Chromatography (HPLC)
•Alcohol production
•Chemical Oxygen Demand (COD)
•Biological Oxygen Demand (BOD)
ENZYMATIC TESTS:

Dehydrogenase Enzymes (DHGs)

Enzymatic tests rely on measurement of either enzyme activity or enzyme

biosynthesis. Enzyme activity is a measure of the catalytic ability. Dehydrogenases
(DHGs) are the enzymes mostly used in toxicity testing. Dehydrogenases oxidize a
substrate by transferring hydrogen to an electron acceptor, common electron
acceptors being NAD+ or FAD. Assay of dehydrogenase activity is conveniently
carried out using oxidoreduction special dyes such as triphenyl tetrazolium
chloride (TTC), methylene blue and resazurin which can also be used as electron
acceptors, changing colour once they are in reduced form. By the reduction of
colourless, water soluble substrate (TTC) by dehydrogenases present in a sample
(e.g. soil), an insoluble product with red colour (triphenylformazan-TPF) is
formed. The TPF can be easily quantified using colorimetric method at the range
of visible light (485 nm). This test however, reflected positive answer only at
neutral range of pH and in presence of calcium carbonate for buffering soil system.
Other enzyme activity tests utilize ATPases, Esterases, Phosphatases, Urease,
Luciferase, Beta- galactosidase, Protease, Amylase, Or Beta-glucosidase. Enzyme
biosynthesis was found to be generally more sensitive to organic chemicals than
enzyme activity. Recently, the inhibition of enzyme (beta-galactosidase,
tryptophanase, alpha-glucosidase) biosynthesis has been explored as a basis for
toxicity testing.
Reduced
TTC DHGs
→ TPF → COLORIMETER
 (colourless, soluble) (red colour,insoluble) (@ 485nm visible light range)

BIOLUMINESCENCE
Intensive industrialization and the use of chemicals in Agriculture and other sectors
have contributed to the release of many toxic compounds into water, air and soil,
which causes many environmental problems. The exposure of living organisms to
toxic levels of pollutants can cause disease to human and animals. The monitoring
and detection of toxic chemicals is very important for the overall safety and
security of humans and all biota on earth. In order to assess these toxic chemicals
in the environment, bioassays and biosensors have been developed.
Bioluminescence refers to the production of light by living organisms.
Bioluminescent microbial biosensors have been used to monitor bioavailable heavy
metals in the environment (e.g. soil, water, industrial effluent, waste water and
stream). Bioluminescent sensing technique used in microbial biosensor is based on
the change in luminescence emitted by living microorganisms in the presence of a
target analyte. For example, using Vibrio fischeri, the light intensity produced is
reduced in the presence of toxic compounds. In bioluminescence, a Luciferin
produces light and a Luciferase(enzyme) allows the light producting chemical
reaction to take place. Luciferase catalyzes the oxidation of Flavin Mononucleotide
(FMNH2) and the long-chain fatty aldehyde (RCHO) by Oxygen with the emission
of blue-green light. The Lux gene codes for Luciferase enzyme and is the most
popular reporter gene in bioluminescent microbial biosensor.

FMNH2 + O2 + RCHO
Luciferase → H + H2O + RCOOH + LIGHT
❑
(Reduced Flavin (Long chain aldehyde)

Mononucletide)
Detection of pollutants using bacterial bioluminescence, e.g Heavy metals,
PAHs etc
Heavy metals such lead, arsenic, mercury, chromium, cadmium etc can be detected
using bacterial bioluminescence. Even Polycyclic Aromatic Hydrocarbons (PAHs),
which are classes of chemicals that occur naturally in coal, crude oil and gasoline
can also be detected using bioluminescence. The bioluminescence inhibition assay
is based on the marine gram negative bacteria Vibrio fischeri or Photobacterium
phosphoreum. The test relies on the change in the bacterial luminescence when the
microorganisms are exposed to toxic chemicals. Example, the Vibrio fischeri and
the Hawaiian Bobtail Squid. V. fischeri lives symbiotically in Bobtail squid.
There is high bioluminescence when V. fischeri is attached to Bobtail squid and no
pollutant encountered. But bioluminescence of Vibrio fischeri decreases when it is
exposed to any flame retardant such as pollutants. Luminescence microorganisms
have been used in the production of several toxicity test instruments. The specific
strain, V. fischeri NRRL B11177, has been used as commercial test kits eg
Microtox, LUMIStox, TOXAlert.
USE OF ATP (Adenosine Triphosphate) IN TOXICITY ASSAY

All cellular activities such as microbial growth, enzyme activities, bioluminescence

all need ATP to function. ATP is found in all living organisms. ATP, the keystone
of all cellular activities, is present in all living cells and is rapidly destroyed upon
the death of the organisms. Therefore, the basis of the ATP toxicity assay is based
on the change in ATP content in the presence of toxic chemicals. The method of
ATP detection is based on the light producing reaction of the Luciferin-Luciferase
enzyme system derived from the fire fly in which light production is proportional
to the amount of ATP present. That is:

Light Production (Fire fly) α Amount of ATP present

Determination of LC50 or LD50

Chemicals can cause a wide range of effects on our health. Depending on how the
chemical will be used many kinds of toxicity tests may be required, since different
chemicals cause different effects. LD (Lethal dose), LD50 is the dose/amount of a
bioactive substance required to kill 50% of a population/group of test animals at a
given time. LC (Lethal concentration) LC50 is the concentration of a bioactive
substance (toxic chemicals such as antibiotics, heavy metals, toxic organic
pollutants e. t. c.) in air or water required to kill 50% of a population at a given
time. LC50 is inversely proportional to toxicity. A substance with a lower LC 50 is
more toxic than one with a higher LC50.

How LC50/ LD50 Tests Are Done:

Pure form of the chemical may be given to the animals by mouth(oral), by

applying on the skin (dermal), by injection at sites such as blood veins (IV-
intravenous), muscles (IM-intramuscular) or into the abdominal cavity (IP-
intraperitoneal). The LD50 value obtained at the end is identified as the LD50 (oral),
LD50 (skin) etc based on the route. In each case, the LD 50 value is expressed as the
weight of chemical administered per kilogram body weight of the animal and it
states the test animal used and route of exposure or administration e.g. LD 50 (oral,
rat)-5mg/kg, LD50 (skin, rabbit)-10mg/kg, LC50 (rat)-1000ppm/4hr, LC50 (mouse)-
5mg/m3/2hr. LD50 (oral, rat)-5mg/kg means that 5mg of the chemical for every
1kg body weight of the rat, when administered in one dose by mouth causes death
of 50% of the test group. For LC50, the concentration is usually quoted as parts per
million (ppm) or milligrams per cubic meter (mg/m3) or in percentage (%).

Determination of LC50 or LD50

Concentraion Number of
of Ni (mg/L) cells (CFU)
0 6.0 x106
0.1 5.7 x106
0.15 5.9 x106
0.2 4.1 x106
0.25 3.9 x106
0.3 2.6 x106
0.35 3.5 x106
0.4 2.8 x106
0.45 2.45 x106
0.5 2.25 x106
LC50= 0.33mg/L
Assignment

Write on Detection of pollutants using bacterial bioluminescence, e.g Heavy

metals, PAHs etc

LECTURER: AJUGWO, GLORIA .C.