Microbiological
examination of a food is required in order to estimate its shelf-life or its
suitability for human consumption. The total viable count or more often, the numbers
of a particular component of the total flora such as moulds in a cereal,
psychrotrophic bacteria in a product to be stored at low temperature, anaerobes
in a vacuum- packed food, or yeasts in a fruit beverage may be needed. Or it
may be required to ensure that a food meets established microbiological
criteria.
The total mesophilic plate count is widely used as an indication of the
microbiological quality of foods unless they are known to contain large numbers
of bacteria as a result of their preparation such as fermented milk and meat
products.
Microbiological analysis
is also required in case of spoilage or foodborne
illness where the presence of a pathogen is implicated. The methods for
determining an estimate of the total mesophilic count are different from those for
identifying the presence of a pathogen, or its isolation for further study.
The isolation of specific
pathogens, which may be present in very low but significant numbers in the
presence of larger numbers of other organisms, often requires quite elaborate
procedures. It may involve enrichment in media which encourage growth of the
pathogen while suppressing the growth of the accompanying flora, followed by
isolation on selective diagnostic media, and finally the confirmatory tests.
Microbiological criteria or the investigation of an outbreak of foodborne
illness thus require the monitoring of certain products for specific pathogens,
but the difficulties in detecting low numbers of pathogens make it
impracticable as a routine procedure.
Indicator
organism
An alternative to
monitoring for specific pathogens is to look for an associated organism present
in much larger numbers – an indicator organism. This is a concept developed
originally for pathogens spread by the faecal-oral route in water. A good
indicator organism should always be present when the pathogen may be present. It
should be present in relatively large numbers to facilitate its detection. It
should not proliferate in the environment being monitored and its survival
should be similar to that of the pathogen for which it is to be used as an
indicator.
Escherichia
coli
is a natural component of the human gut flora and its presence in the
environment, or in foods, generally implies some history of contamination of
faecal origin. In water microbiology E.
coli meets these criteria very well and is a useful indicator organism of
faecal pollution of water which may be used for drinking or in the preparation
of foods.
There are, however,
limitations to its use in foods where there appears to be little or no
correlation between the presence of E.
coli and pathogens such as Salmonella in meat, for example. E. coli cannot may not grow in water but,
it can grow in the richer environment provided by many foods.
Thus, routine microbial examination of food mainly relies on
viable colony count, and examination of fecal streptococci.
v Viable colony count
Viable colony count/Plate count is used to estimate the number of viable
cells that are present in a sample. The most common procedure for the enumeration
of bacteria is the viable plate count.
*
It
relies on bacteria growing a colony on a nutrient medium. A viable cell count
allows one to identify the number of actively growing/dividing cells in a
sample.
*
In
this method, serial dilutions of a sample containing viable microorganisms are
plated onto a suitable growth medium. The suspension is either spread onto the
surface of agar plates (spread plate
method), or is mixed with molten agar, poured into plates, and allowed to
solidify (pour plate method). The
plates are then incubated under conditions that permit microbial reproduction.
*
It
is assumed that each bacterial colony arises from an individual cell that has
undergone cell division. Therefore, by counting the number of colonies and
accounting for the dilution factor, the number of bacteria in the original
sample can be determined.
Drawbacks
*
Traditional
plate counts are expensive in Petri dishes and agar media
*
The
major disadvantage is that it is selective and therefore biased.
*
The
nature of the growth conditions, including the composition and pH of the medium
used as well as the conditions such as temperature, determines which bacteria
in a mixed population can grow.
*
Since there is no universal set of conditions
that permits the growth of all microorganisms, it is impossible to enumerate
all microorganisms by viable plating.
Key
points
*
Selective
procedures for the enumeration of coliforms and other groups can be designed
using selective media/conditions.
*
For
routine laboratory investigations, it is a good and sensitive method of
detecting the presence of a viable bacterium.
*
This
is the basis of the traditional pour plate, spread plate or Miles and Misra
drop plate and is widely used in microbiology laboratories.
*
In the pour plate method, a sample (usually 1
ml) is pipetted directly into a sterile Petri dish and mixed with an
appropriate volume of molten agar.
*
Even
if the molten agar is carefully tempered at 40 – 45oC, the thermal
shock to psychrotrophs may result in them not producing a visible colony.
*
The
spread-plate count avoids this problem and also ensures an aerobic environment
but the sample size is usually limited to 0.1 ml
*
Since
we usually have no idea of how many bacteria are in a sample, it is almost
always necessary to prepare a dilution series to ensure that we obtain a
dilution containing a reasonable number of bacteria to count.
*
Dilutions in the range 10-1(1/10)
to 10-8 (1/100,000,000) are generally used, although with particular
types of samples the range of dilutions can be restricted.
*
For example, for water that is not turbid, the
dilution needed can be 10− 6
The Miles and Misra drop count and spiral plater have
been developed to reduce costs of traditional plating methods.
v Miles–Misra technique
The Miles and Misra
Method (or surface viable count) is used to determine the number of colony
forming units in a bacterial suspension or sample. The technique was first
described in 1938 by Miles, Misra and Irwin.
* The
inoculum / suspension is serially diluted. A calibrated dropping pipette, or
automatic pipette, delivers drops of 20μl on petri dishes containing nutrient
agar or other appropriate medium.
* Three
plates are needed for each dilution series- an average of at least 3 counts are
needed.
* The
surface of the plates need to be sufficiently dry to allow a 20μl drop to be
absorbed in 15–20 minutes.
* Plates
are divided into equal sectors (it is possible to use up to 8 per plate). The
sectors are labelled with the dilutions.
* In
each sector, 20 μl of the appropriate dilution is dropped onto the surface of
the agar (without touching the surface of the agar with the pipette) and the
drop allowed to spread naturally.
* The
plates are allowed to dry before inversion and incubation at 37 °C for 18 – 24
hours (or appropriate incubation conditions considering the organism and agar
used).
* Each
sector is observed for growth, high concentrations will give a confluent growth
over the area of the drop, or a large number of small/merged colonies.
* Colonies
are counted in the sector where the highest number of full-size discrete
colonies can be seen (usually sectors containing between 2-20 colonies are
counted).
* The
following equation is used to calculate the number of colony forming units
(CFU) per ml from the original aliquot / sample
Some
disadvantages are that the rate of absorption of drops onto the surface of agar
depends upon the environmental conditions like temperature and humidity. And, a
skilled microbiologist is needed to perform serial dilution and to follow
aseptic techniques.
v Spiral plater
A
method for determining the number of bacteria in a solution by the use of a
machine which deposits a known volume of sample on a rotating agar plate in a
decreasing amount in the form of an Archimedes spiral. After the sample is
incubated, different colony densities are apparent on the surface of the plate.
By counting an appropriate area of the plate, the number of bacteria in the
sample is estimated.
This
method was compared to the pour plate procedure and the spiral plate method
gave counts that were higher than counts obtained by the pour plate method. The
time and materials required for this method are substantially less than those
required for the conventional aerobic pour plate procedure.
*
The
system is designed so that most of the sample is deposited near the centre of
the plate with a decreasing volume applied towards the edge.
There is a progressive decrease in the concentration of the sample.
*
This
produces an effect equivalent to a dilution of the sample by a factor of 103
on a single plate, thus saving on materials as well as saving the time required
in preparing and plating extra dilutions.
Comparison of raw and analysed spiral plate images
Spiral platers are used
extensively in food safety testing, as well as the pharmaceutical industry. The
spiral plating method can save time and resources by cutting down on additional
dilution and plating steps.
The system is not suitable for all food samples, as particulate material
can block the hollow stylus through which the sample is applied.
Advantages of spiral plating
- Reduces
the number of serial dilutions needed for microbiological testing-3
dilutions obtained in 1 plate
- Reduces
the number of Petri dishes needed-fewer the number of dilutions, fewer
Petri dishes are required.
- The
Spiral distribution enables a reduction of the counting procedure. In most
cases, the count is only needed to be performed on one portion of the
agar.
Fecal Streptococci
The fecal streptococci
and enterococci are members of the genera Streptococcus and Enterococcus. These bacteria are spherical, gram positive,
grow in chains, are facultative anaerobic and ferment sugars. The enterococci and many fecal streptococci
are normal flora of the gastrointestinal tract, where they are present at high
concentrations. However, they can
sometimes spread from the gastrointestinal tract and cause bacteremia, wound
infections, urinary tract infections and endocarditis, more likely in new borns
and in the elderly.
Enterococci are now a
concern in nosocomial infections as well as infections of non-hospitalized
populations because of growing antibiotic resistance, especially to antibiotics
such as vancomycin.
The streptococci and
enterococci are relatively fastidious in their growth requirements because they
have lost the ability to synthesize many essential nutrients. Therefore, they
require several amino acids and the B vitamins to be grown in the laboratory.
Faecal
streptococci are recovered from faeces in significant
numbers. These species are S. faecalis,
S. faecium, S. avium, S. gallinarum, S. bovis, and S. equinus. In
the water microbiology, fecal streptococci are indicators of recent fecal water
contamination.
*
S.
bovis and S. equinus are predominately found
in animals
*
S. faecalis and S. faecium are more specific to the human gut.
Streptococci can be
classified on the basis of Lancefield (carbohydrate of the cell wall) antigens
(A, B, C, D, E-T). Lancefield divided the streptococci into serological groups
based on the presence/absence of certain carbohydrate antigens. Lancefield's
Group D possesses the antigen glycerol teichoic acid.
Streptococci that posses the
Group D antigen are those generally considered to be faecal streptococci—S. faecalis, S. faecium, S. avium, S.
gallinarium, S. bovis, and S. equinus.
The fecal streptococci
and enterococci belong to serogroup D.
Enterococci
The enterococci include S. faecalis, S. faecium, S. durans and related
biotypes. Enterococci
• grow at both 10°C and
45°C;
• survive at 60°C for at
least 30 min;
• grow at pH 9.6;
• grow in the presence of
6.5% sodium chloride;
• reduce 0.1% methylene
blue in milk.
They also
• exhibit resistance to
the antibiotic vancomycin;
• react with
streptococcal Group D antiserum;
• are bile-esculin
positive;
Faecal streptococci along
with total and faecal coliforms, are generally regarded as the most useful
indicators of faecal pollution, and their use has been recommended
internationally.
The enterococcus group (a
subgroup of the faecal streptococcus that includes Enterococcus faecalis, Ent. faecium, Ent. avium and Ent. gallinarum) is a valuable
bacterial indicator for determining the extent of faecal contamination of
recreational surface waters. A direct relationship between swimming-associated
gastroenteritis and enterococci has been established through epidemiological
studies at marine freshwater bathing beaches
Detection of Faecal
Streptococci
Methods for enumeration of fecal streptococci
*
Most
probable number (MPN) method
*
Membrane filtration
*
Plate
count methods
*
Fluorescent
antibody techniques
*
Biochemical
species identification systems
*
DNA-based
identification systems
The
methods most commonly used to assess faecal streptococci in water are the most
probable number (MPN) and the membrane Filltration (MF) techniques. The MPN
technique is applicable primarily to raw and chlorinated waste water and
sediments, and it can be used for fresh and marine waters. The disadvantages of
the MPN technique are that it requires both time and materials, requiring
low-cost labour. The MF method may also be used for fresh and saline samples,
but it is unsuitable for highly turbid waters. Water with high levels of
colloidal or suspended materials can clog the membrane filter pores and prevent
filltration. Moreover, suspended materials cause spreading colonies that may
interfere with target colonies and thereby prevent accurate counting.
The
pour plate technique (PP), is more suitable for enumerating faecal streptococci
densities in polluted waters. The pour plate method is a simple technique
familiar to water bacteriologists; it is less time-consuming than MPN counts
and cheaper, but it is as accurate as membrane filter counts.
Mediums for the growth of fecal streptococci used in a multiple tube (most probable number; MPN)
method, with a membrane filter (MIF) procedure, or by the agar pour plate
technique are
*
Azide
dextrose broth
*
SF
medium (Streptococcus Faecalis medium)
*
BAGG
broth - Buffered
Azide Glucose Glycerol Medium
*
Ethyl
violet azide broth/ (EVA) broth
*
KF
streptococcal medium (Kenner faecal) streptococcus broth
The World Health Organisation recommends the use of
Enterococcus agar for use with pour plates, spread plates, or membrane
filtration techniques for enumerating faecal streptococci and incubation at
44°C for 48 h.
The MPN or multiple tube dilution (MTD) method can be
used to enumerate faecal streptococci .
1.1
Presumptive isolation in azide dextrose broth (AD)
*
The
AD-EVA procedure involves inoculation of dilutions of sample into replicate
tubes of AD broth.
*
Positive tubes exhibit turbidity following
24-48 h incubation at 35-37°C.
*
These
tubes are confirmed by transfer to Ethyl violet azide broth/ (EVA) broth, where
a positive reaction is indicated by the formation of a purple
"button", or dense turbidity, after 48 h incubation at 35-37°C.
*
The
faecal streptococcus count is estimated from the positive EVA tubes using an
MPN table.
*
Various
other broths have been recommended for the recovery of faecal streptococci,
including Kenner faecal streptococcus (KF) broth and Pfizer selective
enterococcus (PSE) broth
*
However,
the AD-EVA method is probably the most commonly used liquid medium system, and
is recommended by WHO.
1.2
Presumptive
isolation in AD broth for 24-48 h is followed by confirmation by streaking a
portion of growth to plates of PSE (Pfizer selective enterococcus) agar that
are then incubated for 24 h at 35°C.
2. Membrane filtration involves drawing a known volume of liquid sample
through a membrane filter with a 0.45 um pore size.
*
Bacteria
present in the liquid are retained on the filter surface and the filter is then
transferred to a selective medium for colony development.
*
The
number of colonies counted on the membrane is taken to be equivalent to the
number of streptococcal cells present per volume of sample.
*
Compared to the most probable number method,
membrane filtration produces results more quickly, allows a larger volume of
sample to be tested, allows colonies to be counted directly, and allows the
immediate biochemical testing of isolated colonies.
*
However,
filtration may not be used for highly turbid samples and is not recommended for
chlorinated effluent.
*
KF
broth/agar (Kenner
faecal streptococcus) was
probably the most commonly used faecal streptococci membrane filtration medium.
*
Faecal streptococci appear on this medium as
pink to dark red colonies after incubation at 35°C for 48 h.
* When
the membrane is overlaid with PSE medium, faecal streptococci colonies can be
distinguished by the presence of dark brown halos indicative of esculin hydrolysis.
3. The bile-esculin test is a biochemical
test performed to differentiate Enterococci and group D Streptococci from
non-group D viridans group Streptococci on the basis of their ability to
hydrolyze esculin.
- Esculin is a glycosidic fluorescent compound, and its hydrolysis
can also be observed by the loss of the fluorescence.
- The
blackening of the medium and loss of the fluorescence demonstrates a positive
test. The lack of color change demonstrates a negative tube test.
· Many organisms are capable of hydrolyzing esculin, but only a few of
them can do so in the presence of bile (4% bile salts or 40% bile.). This
property is utilized to identify organisms of Enterobacteriaceae,
genera Streptococcus and Listeria etc.
· The Bile-esculin test is performed on a selective differential agar;
bile esculin agar, which consists of bile as well as esculin.
· The agar contains different bile salts that inhibit the growth of other Gram-positive organisms and allows the
selective isolation of Enterococci and Group D Streptococci.
· Nowadays, bile-esculin disks are available that are commonly used for
the rapid differentiation between Group D Streptococci and non-Group D
Streptococci.
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