young Whatsapp Call Girls in Vivek Vihar 🔝 9953056974 🔝 escort service
PPT on spoilage (1).pptx
1. CONVENTIONAL AND MODERN
METHODS OF DETECTION OF
SPOILAGE AND CHARACTERISATION
PRESENTED BY:
ASRITHA. R
I MSc. MICROBIOLOGY
GUIDED BY:
DR. BHARATHI S
VICE PRINCIPAL AND HOD
DEPARTMENT OF MICROBIOLOGY
2. INTRODUCTION:
Food spoilage – a metabolic process that causes
foods to be undesirable or unacceptable for human
consumption.
Need for detection:
• to determine its quality.
• to estimate its shelf life.
• to estimate its sustainability for human consumption.
• to confirm that it meets some established microbial criterion.
• to identify the cause of spoilage or the presence of a pathogen
where the food has been implicated in foodborne illness.
• to reduce food loss.
3. for detection of food spoilage:
Numerous methods for detection of spoilage have been
devised with the goals of determining concentrations of
spoilage microbes or volatile compounds produced by
these microbes.
Methods
Conventional
methods
Modern
rapid
methods
Most commonly used, time-consuming
More sensitive and specific than conventional
methods
4. Conventional microbiological assays have been a valuable tool
for specific enumeration of indicative bacteria of relevance to
food and public health, but these culture-based methods are
time-consuming and require tedious biochemical and
morphological identification.
CONVENTIONAL METHODS:
Plate
count
method
Most
probable
number
Direct
microscopic
count
Membrane
filtration
method
Dye
reduction
test
5. Pour plate technique:
Disadvantage: Cells suspended in
the melted agar during preparation
may be heat damaged, due to which
they may not form colonies.
Spread plate technique:
Colonies should be small in size such
that they are clearly distinguishable
and can be counted using a colony
counter.
6. DIRECT MICROSCOPIC COUNT
Pre requisite: Large numbers of cells should be present
in the sample taken.
The microbes should be homogeneously distributed
throughout the culture.
Disadvantage: Cannot distinguishing between living
and dead cells.
7. MOST PROBABLE NUMBER
• Used to estimate the concentration of viable microorganisms in a
sample by means of replicating liquid broth growth in ten-fold
dilutions.
8. SETP 1: PRESUMPTIVE TEST
Compare the number of tubes giving a positive
reaction to a standard chart and record the number of
bacteria present in it.
Incubate all the tubes at 37°C for 24 hrs. If no tubes
appear positive re-incubate up to 48 hrs.
Transfer 0.1 mL sample to the remaining 5 tubes
containing 10 mL single strength medium.
Transfer 1 mL of sample to 5 tubes containing 10 mL
single strength medium
Transfer 10 mL of sample to 5 tubes containing 10 mL
double strength medium.
Take 5 tubes of double strength and 10 tubes of
single strength
Observation Result
Production of gas and
change in the color of the
medium to yellow.
Positive ( proceed to
confirmatory test).
No acid or gas
production.
Sample is considered
microbiologically safe.
9. STEP 2: CONFRIMATORY TEST Some microorganisms other than
coliforms also produce acid and gas from
lactose fermentation. In order to confirm
the presence of coliform, a confirmatory
test is done.
3 mL lactose-broth or brilliant
green lactose fermentation tube
to an agar slant
Incubate
at 37◦C for
24 to 48
hrs
Transfer one loopful inoculum from the
positive results of the presumptive test into:
Check for acid and
gas production
Gram’s staining
10. SETP 3: COMPLETED TEST
Gram’s staining:
Since some of the positive results
from the confirmatory test may be
false, it is desirable to do completed
tests. For this inoculum from each
positive tube of the confirmatory test
is streaked on a plate of EMB or
Endo agar.
12. DYE REDUCTION TESTS:
A group of tests which have been used for
some time in the dairy industry depend on the
response of a number of redox dyes to the
presence of metabolically active micro-
organisms.
The most commonly used dye reduction tests
are MBRT and resazurin reduction test.
13. METHYLENE BLUE
REDUCTION TEST
Principle:
• Methylene blue is a redox indicator, that loses
its color under the absence of oxygen and is
thought to be reduced. The depletion of
oxygen in the milk, and the production of
reducing substances in the milk is caused by
enhanced rate of bacterial metabolism. The
dye reduction time refers to the microbial load
in the milk and the total metabolic reactions of
the microorganisms.
14. RESAZURIN REDUCTION
TEST:
• In RRT, at a fixed period of time, specific shade of
color and its intensity is measured.
COLOR GRADE OF MILK
BLUE EXCELLENT
LIGHT BLUE VERY GOOD
PURPLE GOOD
PURPLE PINK FAIR
LIGHT PINK POOR
PINK BAD
WHITE/COLOURLESS VERY BAD
15. LIMITATIONS
Conventional methods are usually inexpensive and simple, but
these methods can be time consuming.
Laborious as they require the preparation of culture
media, inoculation of plates and colony counting.
May be limited due to their low sensitivity.
False results may occur due to viable but non culturable pathogens.
To overcome these limitations, modern methods that are rapid
and much more sensitive have been developed.
17. DIRECT EPIFLUORESCENT FILTER TECHNIQUE
• It is an improved version of the membrane filter technique.
• Employs fluorescent dyes and fluorescent microscopy.
Diluted food
homogenate
Filter
through 5
µm nylon
filter
Collect the
filtrate
Treat with 2
ml Triton X-
100 + 0.5 ml
trypsin
Incubate for
24-48 hrs
Pass the
filtrate
through 0.6
µm
nucleopore
polycarbonate
membrane
Stain the
filter with
acridine
orange
Enumerate the
stained cells
using
epifluorescence
microscopy
18. IMPEDANCE
o Principle:
Bacteria growing actively in a culture medium
produce positively or negatively charged end
products that cause an impedance variation of the
medium.
o Impedance Variation number of bacteria
o Detection time (DT) 1/ log no. of bacteria in the
sample.
21. DNA/RNA METHODOLOGY
Release of nucleic
acids through cell
lysis
Denaturation in
case of DNA to
give ssDNA (heat
treatment)
Adsorption of
denatured nucleic
acid onto a
memebrane
Fixation by using
heat or alkali
treatment
Treatment with
blocking agent
Incubate with
labelled probe
Wash off
unadsorbed
probes
Measurement of
labelled probe
23. ADVANTAGES
AND
LIMITATIONS
OF USING
RAPID
TECHNIQUES
ADVANTAGES
• Rapid
• Sensitive
• Specific
• Time efficient
• Labour saving
• Reduces human errors to a great extent
LIMITATIONS
• Enrichment of sample, culturing is the time-
consuming step
• Requires cell lysis or heat denaturation to
expose Ag or targets.
24. CHARACTERISTICS OF SPOILAGE
SPOILAGE SIGNS
• Souring- due to production of acid. Ex. Sour milk from the production of lactic acid
• Gas formation- meat becomes spongy, swelling or bubbling of packages and cans
• Odor- due to breakdown of proteins (putrefaction).
• Sliminess- primarily due to surface accumulation of microbial cells,
also due to tissue degradation
• Discoloration- mold on bread (blue or green molds on citrus fruits and cheese)
SPOILAGE IN MILK
• Ropiness and sliminess can occur in milk and cream.
• Ropiness is usually caused by the slimy capsular material from bacterial cells.
25. PROTEOLYSIS
• Production of bitter flavor due to hydrolysis of milk proteins by
microorganisms.
SPOILAGE OF MEAT AND FISH
• Autolysis, oxidation or bacterial activity
• Surface slime due to action of Pseudomonas, Moraxella,
Lactobacillus etc.
• Color change to green, brown or grey.
• Change in fats due to rancidity, lipolysis, oxidation of fats
• Off odors and off tastes
26. CONCLUSION
• Methods to detect foodborne
pathogens have relied on culture
based techniques that are labour
intensive and time consuming.
• To overcome these limitations, new
techniques that are faster and more
sensitive have been developed.
• These assays include miniaturized
biochemical tests, specialized media
and substrates, and DNA- and Ab-
based detection assays.
27. REFERENCE
• Adams M. R. and Moss M. O. Food Microbiology (3rd edition).
RCS publishing. Pg 370-394
• Montville T. J and Matthews K. R. Food Microbiology-An
introduction (2nd edition). ASM press. Pg. 65-76.
• Jay J. M., Loessner M. J., and Golden D. A. Modern Food
Microbiology (7th edition). Pg. 217-225
• Senan S., Malik A. R. K. & ShilpaVij. Food and Industrial
microbiology. Pg. 117-121
• Doyle M.P, Beuchat L.R, Montville. T. J. Food Microbiology-
Fundamentals and Frontiers (2nd edition). ASM press. Pg.775-793
28. PREVIOUSLY ASKED QUESTIONS
• Explain the conventional and modern methods used for the
detection of spoilage and characterization [15 M]
• Methylene blue reductase test [5 M]
Pour plate tech. – colonies that grow inside the media are smaller
Spread plate tech. – single living cell divides to form a colony.
Therefore, each bacterium represents a colony forming unit.
Measured as cfu/ml.
cfu*dilution factor
Haemocytometer calculation-
Inference: absence of gas and failure to demonstrate gram negative non spore forming bacilli – negative test.
One plate is incubated at 37 C and other at 44.5 C for 24 hrs – detection for thermotolerant E.coli.
Observe for typical colonies- green metallic sheen at 37 C and colonies formed o C plate is confirmation for presence of E.coli
Redox dyes: anilinic acid, diphenylamine
Redox potential: it is a measure of the ease with which a molecule will accept electrons, which means that more positive the redox potential, the more readily a molecule is reduced.
Redox potential:
Resazurine dye : blue- +0.3V
Pink : +0.2 V (resorufin)
Coloruless : +0.1V (dihydroresorufin)
Color change detected using Lovibond color compactor and standard resazurin disc
Impedence: ia a measure of the opposition to the electrical flow.
it is similar to resistance.
bactometer A device for the estimation of bacterial contamination within a few hours, based on measuring the early stages of breakdown of nutrients by the bacteria through changes in the electrical impedance of the medium.
Used in detecting milk spoilage..
Rapid mATP bioluminescence assay. The bioluminescence assay was performed using a microluminometer NHD model 3560 and PROFILE-1 reagent kit (New Horizons Diagnostic, Columbia, Md.). The reagent kit includes the Filtravette, somatic cell–releasing agent, bacterial-releasing agent, and luciferin-luciferase. The swab in the sample vial was first vortexed for 30 s. The suspension was then transferred using a sterile pipette tip to the Filtravette, a combined device of filter and cuvette with a pore size of 0.45 m, underlined with a blotter paper to absorb the filtrate. The suspension was then pushed through the Filtravette by a positive pressure device (a 3-ml syringe with o-ring attached to the top). The assay volumes were between 0.2 and 1.0 ml, based on the filterability of the suspension. After the desired volume was passed through the Filtravette, three drops of somatic cell–releasing agent were added and the mixture was pushed through the Filtravette by the positive pressure device. Three more drops of somatic cell–releasing agent were added and pressure filtered to ensure the removal of interfering substances, free ATP, and somatic cell ATP. The Filtravette was then placed into the drawer slide of the microluminometer. Two drops of bacterial releasing agent were added into the Filtravette to extract the microbial ATP. Immediately after the addition of the bacterial releasing agent, 50 l of luciferin-luciferase was added and mixed by aspirating the fluid up and down three times. The drawer slide was closed immediately; light emission was measured with integration over 10 s. Bioluminescence from mATP was measured directly from the microluminometer digital readout as relative light unit (RLU)
Used in dairy industry
Used in meat and fish industries.
a major drawback of the hybridization assays is their lack of sensitivity, which limits the use of these analyses to populations of cells or genes occurring in relatively high numbers in samples. For this reason, hybridization assays are currently mainly used for culture confirmation rather than direct detection and identification.
FISH assays have been developed and used to detect at family, genus and species level Staphylococcus spp., Listeria spp., Campylobacter spp., Salmonella spp. and E. coli
Salmonella spp., Listeria spp., Listeria monocytogenes, Campylobacter jejuni, Cryptosporidium parvum, E. coli
PCR tests can detect spoilage organisms in beer .
Thermus aquaticus
Milk- Micrococcus, some Enterococcus, Streptococcus, some Lactobacillus, and spores of Bacillus and Clostridium.
Fungi - Aspergillus, Cladosporium, Candida spp.
Meat- Pseudomonas, Acinetobacter, Alcaligens, Micrococcus, Sarcina, Leuconostoc, Lactobacillus, Proteus
Fish- Bacillus, Clostridium, Escherichia, Micrococcus, Proteus, Sarcina
