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MiCROBIAL ANALYSIS OF FOOD & DRINKING WATER Public Health

Microbiology (ML 2214)

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 MiCROBIAL
ANALYSIS OF FOOD &
DRINKING WATER
Public Health Microbiology
(ML 2214)
Dr. Manosha Perera
(BSC., MSc., PhD)
 OUT LINE
1.Microbial Analysis of
Food
2. Microbial Analysis of
Drinking water
1.Microbial Analysis of Food
 Detection of Indicator Organisms & Detection
of Pathogens.
 Indicator organisms are typically used to
demonstrate the potential presence or
absence of groups of pathogens
 The use of indicators is attractive because it
reduces the complexity and cost of analyzing
sludges or environmental media (soil, food,
water, air) for individual pathogens
 Indicator microbes are generally
selected due to
1) They are initially abundant in the matrix to be
assayed.
2) A relatively rapid, accurate, and cost effective
analytical method for enumerating the indicator
exists or can be readily developed.
3) A reasonably strong correlation exists between
the presence/absence of the indicator and a
particular pathogen or group of pathogens.
4) The strength of the correlation will determine
the effectiveness and accuracy of the indicator as
a measure of pathogen occurrence.
 OBJECTIVES
 General Objective
To understand the basic analytical
methods for identification and
quantification of microorganisms
in food and water samples.
 Specific Objectives
1. To understand/perform Total Bacterial Count/Aerobic
Plate Count of Food Samples.
2. To know/presumptive Coliform, confirmation of
coliforms, E.coli Counts by Most Probable Number
(MPN) method & Enterobacteriaceae count in Food
Samples.
3. To understand/perform the Isolation & identification of
Salmonella species from food samples
4. To understand/perform the Isolation & identification of
Staphylococcus aureus from food samples.
5. To learn/perform the steps involved in isolation and
identification of B.cereus in food samples
 Importance
 Food safety is utmost importance in public health, travel&
trade
 Some microorganism: undesirable changes/ alter esthetic
quality in food or can be primary vehicle for transmission of
foodborne pathogens.
 Analytical Food Microbiology : Indicators, pathogens & spoilage
organisms
 Hygienic Quality of Food: Aerobic Plate Count and Faecal
Indicator organisms such as E.coli, coliforms & faecal
Streptococci.
 Presence of Indicator organisms : exceeding the numbers
certain guide value (Sri Lankan Standards for different food
types) : unhygienic handling of food including the possible
presence of pathogens.
 1.Total Bacterial Count/Aerobic Plate
Count of Food Samples
 Total Bacterial Count/Aerobic Plate Count/Standard
Plate Count/Total Viable Count : total number
of bacteria able to grow in an aerobic environment in
moderate temperature.
The total bacteria count is one of the key indicators
in the field of hygiene management.
It indicates how many microorganisms (live) are
present in a sample.
Monitoring the total bacteria count is necessary,
because the number of microorganisms shouldn’t
exceed certain guide values.
 Overview
 This method assumes that the microbial cells
present in a sample when mixed with agar medium:
visible separate colonies
 By mixing decimal dilutions of the food sample
homogenate with the agar medium
After incubation of the plates at 300C for 72 hours
the number of mesophilic aerobic bacteria/gram of
food sample is calculated by the number of colonies
obtained in selected plates at levels of dilutions
produced significant results.
 Inherent limitations of APC
 Clumping of colonies / absence of separate colonies:
Microbial cells frequently occur as clusters, chains or
pairs in food may not well distributed irrespective of the
mixing & dilution techniques.
 Each colony : may arise from a single cell or from groups
of cells and colony count may not reflect the actual
number of viable bacteria in the food.
 Failure of forming visible colonies on the agar medium:
unfavourable conditions including adverse T0,
undesirable [O2], poor nutrition, cells are damaged and
not multiply (not in log phase of growth).
 Requirement
1.1 Apparatus & glassware
a) Petri dishes(90-100) glass or plastic
b)Pipettes 1.5 and 10 ml, graduated (total flow)
c)Water bath ,45 ± 10C
d)Incubator, 30 ± 10C
e)Colony counter
f) Sterile spatula
1.2 Culture media & diluent
a)0.1% Buffered peptone water (0.1% BPW)
b) Plate count agar
 1.3 Test Procedure
1.3.1 Preparation of food homogenate
 Weigh 10g of the mixed sample aseptically
using a sterile spatula into a sterile blender jar
or into a new stomacher bag
 Add 90ml of 0.1% Buffered peptone water.
 Blend the food at a speed of 15000-
20000rpm for 90 seconds OR
 Mix in the stomacher for 20 seconds.
 1.3.2 Making dilutions
 Mix the food homogenate by gentle shaking & pipette
1.0 ml & dispense into tube containing 9 ml of the 0.1%
Buffered peptone water (10-1)
 Mix carefully by aspirating 10 times with a fresh pipette
and transfer 1.0 ml from the first dilution with the same
pipette to second dilution tube containing 9ml 0.1% BPW
(without touching). Mix well by shaking 25 times in a
30cm arc to obtain 10-2 dilution.
 Repeat the procedure using the 3rd & 4th tube or more
until the required number of dilutions are made.
 Shake all dilutions carefully.
 1.3.3 Pour Plating
 Pipette 1ml of the homogenate from each dilution into each of
the appropriately marked duplicate petri dishes.
 Pour 15 ml of PCA (Plate Count Agar) into each petri dish, kept
at 45 ± 10C in a water bath.
 The time passing between the preparation of initial
suspension & the pouring of PCA into Petri dishes should not
exceed 30 minutes.
 Mix the sample dilution and agar medium thoroughly and
uniformly by alternate rotation and back-and-forth motion of
plates on flat level surface.
 Let agar solidify
 Invert solidified petri dishes, and incubate promptly for 48 ± 2 h
at 35°C. Do not stack plates when pouring agar or when agar is
solidifying
 1.3.4 Calculation of Results
 Counting : Duplicate plates containing 15-300
colonies, at two consecutive dilutions (e.g. 1:
10 and 1: 100 diluted samples).
 It is necessary that at least one of these
plates contain ≤15 colonies.
 Calculate the number of microorganisms
(N)/ml or gram of product using the equation
 1.3.4 Calculation of Results
Calculate the number of microorganisms (N)/ml or
gram of product using the equation

where:
N = Number of colonies per ml or g of product
∑ C = Sum of all colonies on all plates counted
n1 = Number of plates in first dilution retained
n2 = Number of plates in second dilution retained
d = Dilution from which the first counts were obtained
 Example
Colonies in 1:100 Colonies in 1:1000
232, 244 33,28
Colonies in 1:1000

= 537/0.022
= 24,409
≈ 24,000
2.4 X 104cfu / g or ml of food
 1.3.4 Calculation of Results
 Round the results calculated into two significant
figures.
 When the number to be rounded is five with no
further significant figures, round the number to give
an even figure immediately to the right. For example
28500 are rounded to 29000. Moreover, 11800 are
rounded to 12000
 21136 rounded immediately to the left to give an
even number 21000 & 23463 rounded to 23000.
 1.3.5 Reporting Results (in non ideal
conditions)
Report the results as the number of microorganisms/ml or per gram
expressed as a number between 1.0 and 9.9 multiplied by 10x,
where X is the corresponding power of 10.
 No colonies: If the two dishes corresponding to the test sample
(liquid products) or the initial suspension (other products)
containing no colonies. Report as follows.
REPORT AS APC = Less than 1X 101 /g or/ml
 Less than 15 colonies: When there are < 15 colonies, calculate the
arithmetic mean “m” of the colonies counted in both dishes.
Report as APC = “m”X dx /g or/ml
 When only one dilution was in appropriate range ,average count /g
for dilution was calculated & reported as APC = “m”X dx /g or/ml
 More than 300 colonies : If no plate procuresses < 300 colonies
calculate the results for the plate with lowest number of
microorganisms ‘m’ X dx/ml or/g
dx Reciprocal of the dilution factor
2. Coliform, faecal coliforms, E.coli Counts by Most
Probable Number (MPN) method &
Enterobacteriaceae count in Food Samples.

2.1 Method for the Presumptive Coliform Count

MPN method (SLS 516: part 3. 1982)
This method based on a statistical technique for
estimating the most probable number of
bacteria per specific unit of material under test,
using acid & gas production in Mac Conkey
broth or Lauryl Sulphate Tryptose broth
followed by confirmation of gas production in
Brilliant Green Lactose Bile broth each being
incubated at 370C for 24-48 hrs.
 2.1 Method for the Presumptive Coliform
Count MPN method (SLS 516: part 3. 1982)
2.1.1 Apparatus & glassware
a) Test Tubes (18 mmx180 mm)
b) Durham tubes ( 10mmx 75 mm)
c) Pipettes 1ml and 10 ml
d) Incubator
e) Water Bath
f) Blender or Stomacher blender
 2.1 Method for the Presumptive Coliform
Count MPN method (SLS 516: part 3. 1982)

2.1.2 Culture media & Reagent

a) Mac Conkey broth or Lauryl Sulphate broth
b) 0.1% Buffered Peptone Water (BPW)
c)Brilliant Green Lactose Bile Broth (BGLB)
d) Indole medium (Tryptophan or peptone
broth)
e) Kovac’s or Ehrlic’s reagent
 2.1.3 Procedure
2.1.3.1 Preparation of food homogenate &
dilutions are described as previously in 1.3.1.
 2.1.3 Procedure
2.1.3.1 Inoculation
 Inoculate each of 3 tubes of double strength of
MacConkey broth (containing inverted Durham
tubes) with 10ml of the food homogenate (10-1).
 Inoculate each of 3 tubes of single strength of Mac
Conkey broth (containing inverted Durham tubes)
with 1ml of the food homogenate (10-1).
Inoculate each of 3 tubes of single strength of Mac
Conkey broth (containing inverted Durham tubes)
with 1ml of the food homogenate (10-2).
Incubate all tubes at 37 ± 10C for 24-48 hrs.
 2.1.3.2 Reading of enrichment tubes
(Presumptive Test)
 Record tubes showing gas production & colour change
(Purple to Yellow) after 24 hrs, and re =incubate negative
tubes for further 24hrs, then record tubes showing acid & gas
production.
 2.1.3.3 Calculation (MPN)
 Note the MPN corresponding to the number
of POSITIVE tubes from the MacGrady’s Table.
No: of positive Tubes
0.1 0. 01 0.001 MPN/g
0 0 0 <3.0
0 0 1 3.0
0 1 0 3.0
3 3 2 1100
3 3 3 >1100
2.2 Confirmation of Coliforms & E.coli
 Transfer a loopful from each Positive tube of
Mac Conkey broth to two separate sets of
BGLB and Tryptophan or peptone broth.
 Incubate one set at 37± 10C for 48 hrs
 The formation of gas & positive indole test
confirms the presence of bacteria
 2.3 Confirmation of Faecal coliforms
 Gas production BGLB incubated at 44 ± 10C
for 48 hrs confirms the presence of Faecal
Coliforms.
 2.4 Confirmation of E.coli
Incubate The other set at 44 ± 10C for 48 hrs
 The formation of gas in Durham tubes &
positive Indole test (red ring) confirms
E.coli.(This is a modification of Eijkman test).
 2.5 Enterobacteriaceae Count
 Ability to grow in the presence of bile salts & produce acid from
glucose is used to grow family Enterobacteriaceae in Violet
Red Bile Glucose agar.
 Bile salts and crystal violet selectively inhibit Gram positive
bacteria.
 Glucose fermenters (who could tolerate bile salts) : Red purple
colonies with red purple halos (bile precipitation) in the
presence of Neutral red pH Indicator.
 2.5 Enterobacteriaceae Count
2.5.1 Apparatus & glassware
a) Petri dishes 90 -100 mm, glass or plate
b) Pipettes 1ml, 5 ml & 10 ml (graduated total-flow)
c) Water bath 45 ± 10C
d) Incubator 42 ± 10C
2.5.2 Culture media & diluents
a) 0.1% Buffered peptone water (0.1% BPW)
b) Violet Red Bile Glucose agar
 2.5 Enterobacteriaceae Count
2.5.3 Test Procedure
Preparation of food homogenate & dilutions are described
under 1.3.1 & 1.3.2.
2.5.4 Inoculation
• Transfer 1ml of aliquots of each dilution to 9 cm petri dishes, duplicate
plates in each dilution.
• Pour 15 ml of the medium (kept at 45 ± 10C in a water bath). Gently swirl
the plates 3 times clock wise & 3 times anti clock wise.
• After the medium has solidified overlay with 10 ml of the same medium
(violet red bile glucose agar) & allow to solidify.
• Invert the petri dishes & incubate at 420C for 18hrs, 320C for 24-48 hrs or
40C for 10 days depending on the groups of Enterobacteriaceae to be
recovered.
• For routine purpose, incubation at 370C for 24-48 hrs is satisfactory.
 2.5 Enterobacteriaceae Count
2.5.5 Colony Morphology
 Round, purple to red, 1-2 mm in diameter and surrounded by purple haloes. The
colour of colonies can vary depending on the source of medium.
 Generally 1-2 mm in diameter
 Counts can be expressed as N/ml or g of product. Highest dilution which
contains 15-150 purple red colonies .

For example: 80 colonies in 10-3, 120 colonies in 10-3

10-3

200 /2 x 103

100 x 103 cfu/ g

1.0 X 10 5 cfu/g
 Summary
 Food samples : Aerobic plate count & faecal indicator
organisms such as E. coli, Coliforms &
Enterobacteriaceae
 These indicator microorganisms are presumed to
indicate unhygienic handlying including possible
presence of enteric pathogens.
 The Aerobic plate count: One of the most useful,
basic indicator of the microbiological quality of food.
A high viable counts often indicates : Contaminated
raw material, unsatisfactory sanitation, time T0 abuse
during production or storage or a combination of
these.
 Summary
 Presumptive Coliform count: MPN method using
MacConkey broth or Lauryl Sulphate Tryptose broth.
 Gas production and colour change (Purple to Yellow)
after 24-48hrs indicate positive results.
 Confirmation of Coliforms performed by growth & gas
production in BGLB and positive Indole test (incubated
at 37± 10C for 48 hrs.
 Confirmation of faecal coliforms by growth & gas
production at 44 ± 10C for 48 hrs.
 Confirmation of E.coli gas production at 44 ± 10C for 48
hrs and positive Indole test.
 Identification & enumeration of Enterobacteriaceae
purple to red colonies with purplish halos in Violet Red
Bile Glucose agar.
 3. Isolation & identification of
Salmonella species from food samples
 Gram negative, non spore forming rods &
members of the family Enterobacteriaceae
 During the conduct of outbreak investigation,
Initially Salmonella may be isolated from clinical
specimens as well as incriminated food.
 Eggs, egg products, milk, dairy products, raw or
cooked meat, meat products,fruits,vegetables
noodles, macaroni & spaghetti are more
susceptible to contaminated with this bacterium.
 3. Isolation & identification of
Salmonella species from food samples
Salmonella causes two kinds of illness:
(1) Gastrointestinal illness, which causes nausea, vomiting, diarrhea, cramps,
and fever, with symptoms generally lasting a couple of days and tapering off
within a week. In otherwise healthy people, the symptoms usually go away
by themselves, but long‐term arthritis may develop
(2) Typhoidal illness causes high fever, diarrhea or constipation, aches,
headache, and lethargy (drowsiness or sluggishness), and, sometimes, a
rash. It’s a very serious condition; up to 10% of people who don’t get
treatment may die.
(3) The Typhoidal illness usually is associated with sewage‐contaminated
drinking water, or crops irrigated with sewage‐contaminated water.
(4) Some pets, like turtles and other reptiles, and chicks, can carry Salmonella,
which can spread to anything that comes into contact with the pet
 3. Isolation & identification of
Salmonella species from food samples
3.1 Equipment & materials
1. Balance
2. Stomacher
3. Sterile stomacher bags
4. Sterile Spatula
5. Autoclave/Oven
6. Drying cabinet
7. Incubators controlled at 37 ± 10C and 42 ± 10C
 3.2 Media and Reagents
1. Buffered Peptone Water (BPW)
2. Modified Semisolid Rappaport Vasiliadis
(MSRV) agar
3. Xylose Lysine Desoxycholate (XLD) agar
4. Salmonella -Shigella (SS) agar
5. Kligler Iron Agar (KIA) or Triple Sugar Iron Agar
(TSI)
6. Lysine Indole Motility (LIM) Medium
7. Salmonella Antisera
 3.3 Methodology
 25g of the sample is transferred using sterile spatula
into a sterile stomacher bag and 225ml of 1% BPW is
poured in- Pre- enrichment
 Bag is homogenised for 30 seconds & incubated at
370C overnight.
Then 3-4 drops of incubated broth is placed on the
surface of MSRV(enrichment) agar plates and
incubated at 420C for 18 hrs.
Following day, a loopful of suspected growth is plated
on SS agar & XLD (selective agar plates) and incubated
at 370C overnight.
 3.3 Methodology
 Instead of MSRV Selanite Cystine broth and
Tetra Thionate broth can be used.
 More than one enrichment culture may yield
good results
 Mannitol Lysine Crystal Violet Brilliant Green
(MLCB) medium, Desoxycholate Hydrogen
Sulphide Lactose (DHL) medium, Brilliant
Green (BG) are can be used as selective media
as well.
 3.4 Identification
 Suspected growth in MSRV Medium- Cloudy,whitish,thick
surface growth.
 3.4 Identification
SS agar: Pale colonies with black canters

 XLD agar: Pink colonies with black centre

 3.4 Identification
 Suspected colonies with characteristic colony
morphology are inoculated into KIA/TSI and LIM tubes
with relevant biochemical profiles suggestive of
Salmonella species.

Alkaline slant

Marked H2S production

Acid but

Typical Kligler pattern of food poisoning Salmonella species

 3.4 Confirmation
LIM : Lysine positive, Indole negative and
Motile
 Salmonella species is confirmed by sero
typing with the panel of Salmonella antisera.
 4.Isolation & identification of
Staphylococcus aureus from food samples
 Gram positive coccus which forms irregular grape like clusters.
 The presence of live organisms in food, especially in high counts :
unsuitable for human consumption as it may produce heat stable
enterotoxins.
 Toxin can with stand heating at 1000C for 30 minutes. The elimination of
live organisms does not ensure safety of food : Toxin can persist in it.
 S.aureus: lives as a commensal on skin, mouth & nose
 Can easily contaminate food via the hands of food handlers. and then the
food is not properly refrigerated. Other sources of food contamination
include the equipment and surfaces on which food is prepared.
 Easily destroyed by heating, drying and other food processing methods.
 Hence fruits and vegetables eaten raw are most liable to be contaminated.
Salads such as ham, egg, tuna, chicken, potato, and macaroni. Bakery
products, such as cream-filled pastries, cream pies, and chocolate éclairs
 4.Isolation & identification of
Staphylococcus aureus from food samples
These toxins can cause nausea, stomach
cramps, vomiting, and diarrhea. In more severe
cases, the toxins may cause loss of body fluid
(dehydration), headache, muscle cramps, and
temporary changes in blood pressure and heart
rate. The illness usually is intense, but normally
lasts from just a few hours to a day.
 The toxins are fast‐acting; they cause symptoms
within 1 to 7 hours after contaminated food is
eaten
 4.Isolation & identification of
Staphylococcus aureus from food samples
4.1 Equipment & materials
1. Balance
2. Stomacher
3. Water bath
4. Incubator controlled at 37 ± 10C
5. Sterile stomacher bags
6. Apparatus for sterilization- Hot air oven/Autoclave
7. Petri dishes- glass or plastic, 90-100mm or 140mm
8. Delivery pipettes- 1ml, graduated in 0.1ml
9. Glass spreaders
 4.Isolation & identification of
Staphylococcus aureus from food samples
4.2 Media & reagents
1. Peptone water0.1%
2. Baired Parker medium
3. Rabbit/Human plasma
4. Blood agar
 4.3 Methodology
 10g of the sample is measured into a sterile
stomacher bag or into a sterile blender jar.
 Sample is homogenized for 30 seconds to 1
minutes using a stomacher or a blender.
 Additional dilutions of 10-2, 10-3 or more are
prepared as required.
 4.3 Methodology
 0.1 ml from each dilution in duplicate place
on Baired Parker Medium (BPM) and spread
evenly on the surface using glass spreaders.
 Plates are allowed to dry in RT0 for about 15
minutes and are incubated at 37 ± 10C for
48hrs.
 Plates with a colony count of 15-150 colonies
are preferred for counting.
 4.3 Identification
 Five typical and five atypical colonies are
selected if the sample is a dairy product,
otherwise 10 typical colonies are selected.
 Colony appearance on BPM: appear as black,
shining, convex 1.0-2.5mm in diameter. With
an opaque zone and a zone of clearing around
the colony.
 4.3 Identification
 Baired Parker introduced this complex medium in 1962 to
over solve the problem of recovering damaged S.aureus from
foodstuffs. The medium is highly selective due to potassium
tellurite & lithium chloride.
 Tellurite inhibits most coliforms and is reduced by S.aureus to
telluride to give typical black colonies.
 Two typical reactions of S.aureus can be detected by the egg
yolk in the medium.
 Lecithinase production- an opaque zone is produced around
the colony
 Lipase production – a zone of clearing is produced outside the
opaque zone.
4.4 Identification and confirmation
 Selected colonies are sub cultured on Blood
Agar and incubated at 370C Overnight.
 Colony appearance on BA: Characteristic
colonies are usually β haemolytic, convex,
regular margined, golden yellow/creamy
white/buff coloured.
 Coagulase test is done for
Characteristic colonies
4.5 Enumeration of S.aureus
S.aureus is reported as number of colonies
(X)cfu per gram of food using following
equation.
n1- Number of colonies counted in the first plate (Plate 1)
n2- Number of colonies counted in the second plate( Plate 2)
n - Number of colonies positive for coagulase test
N- Number of colonies taken for coagulase test
D- Dilution factor of the BPM plate
 4.6 Toxin detection
 Enterotoxin (ET) detection is required when
the colony count is > 1.0 X 106/g .
 Follow the manufacturer’s instructions
carefully when performing the test.
 Latex agglutination & ELISA methods are
available for detection of S.aureus
enterotoxin.
 6. Isolation and identification of
B.cereus in food samples
 Gram positive,facultatively anaerobic, motile, spore forming Bacillus.
 Two distinct types of food poisoning syndromes as well as other
manifestations of Pathogenicity.
 Many raw & processed food types such as meat and meat products, fish,
poultry, milk and dairy products, cereals (particularly rice) and cereal
derivatives, starches, herbs and spices.
 Farinaceous foods such as Chinese style cooked rice (fried rice)and other
foods such as spaghetti, noodles, macaroni, potato salads as well as cheese
products, pastseurized cream, eclairs and reconstituted formulas : vehicles
in emetic type food poisoning outbreaks.
 Proteinacious food such as casseroles, sausages and other cooked meat
and poultry dishes, pastries, salads, meats and vegetable soups,
occasionally fish,milk and ice cream have been incriminated for diarrheal
type of food poisoning outbreaks.
 B.cereus group
 B. cereus - motile
 B.mycoides- Non motile
 B.thuringiensis - motile
 B. anthracis – Non motile
 6. Isolation and identification of
B.cereus in food samples
6.1 Equipment and material
1. Balance
2. Stomacher & sterile stomacher bags
3. Incubator controlled at 37 ± 10C and 460C
4. Flat bottomed flasks (500 ml & 1000ml)
5. Measuring cylinders ( 100ml & 500ml)
6. Petri dishes
7. Pipettes 10ml & 1 ml
8. Tubes with screw caps
9. Grease Pencil or marker pen
10. Sterile Spatula
 6.2 Media & reagents
1. Bacillus cereus selective agar base- Polymyxin Egg yolk
Mannitol Bromothymol Blue Agar (PEMBA)
2. Bacillus cereus selective supplement
3. Egg Yolk Emulsion
4. 0.1% peptone water
5. Reagents for Gram stain
6. 5% w/v malachite green
7. 0.3% w/v Sudan black in 70% ethanol
8. 0.5% w/v carbol fuchsin
9. Reagents for nitrate reduction test
 6.3 Methodology
10g of food is aseptically weighed into a sterile
stomacher bag and 90 ml of 0.1% peptone water
is added.
 This is homogenized for 30 seconds in stomacher
(This is the 10=1 dilution).
 Then 1 ml of initial suspension is transferred into
a tube containing 9ml of sterile diluent.
 This is mixed well by shaking 25 times in a 30cm
arc to obtain 10-2 dilution.
 Similarly further dilutions are prepared upto 10-6
 6.3 Methodology
 B.cereus selective agar plates are prepared
according to the manufactures instructions.
 From each dilution portions of 0.1ml (100 µl) of
are transferred into two well dried B.cereus
selective agar plates.
 Then the inoculum is spread gently & evenly over
the surface of plates with a spreader.
 Plates are incubated at 350C overnight.
 Plates are left for further 24hrs at room
temperature in order to detect all the B.cereus
colonies.
 6.4 Enumeration of B.cereus
(Presumptive plate count)
 Typical colonies of B.cereus are crenated, about 5mm in
diameter, peacock blue in colour (mannitol is not utilized) and
surrounded by a zone of egg yolk precipitate of the same
colour (lecithinase activity which hydrolyses lecithin of egg
yolk). B.thuringiensis has the similar colony morphology.
 6.4 Enumeration of B.cereus
(Presumptive plate count)
 Plates that contain an estimated number of
15-150 typical colonies are selected.
 Typical colonies in both plates with the
highest dilution are counted and 10 colonies
are subjected to preliminary & completed
identification tests as outlined below.
 Based on the results of these tests the final
count of B.cereus (cfu)/g of food is calculated.
Calculation
 6.5 Confirmation of B.cereus by
Morphological tests
6.5.1 Gram stain : Short to long chains of Gram
positive bacilli, spores are ellipsoidal, central
to sub terminal and did not swell the
sporangium.
 6.5.2 Rapid confirmatory staining
procedure
 Stain is done from typical colonies of B.cereus
 Smears are prepared from center of 1 day old
colonies or from the edges of 2 day old colonies.
Smears are dried and fixed with minimal heating.
 Smears are flooded with aqueous 5% w/v
malachite green, heated with a flaming alcohol
swab until team rises and left for 2 minutes
without reheating.
 Then they are washed in running water & blotted
dry.
 6.5.2 Rapid confirmatory staining
procedure
 Subsequently flooded with 0.3% w/v Sudan black
in 70% alcohol and left for 15 minutes.
Next slides are washed with xylene from a wash
bottle for 5 seconds.
 Slides are blotted dry with a filter paper.
 Lastly slides are flooded with aqueous 0.5% w/v
carbol fuchsin for 20 seconds, washed under tap
water
 Blot dried and examined under the oil immersion.
Characteristic appearance of B.cereus
 Vegetative cell is 4-5 µ long and 1.0-1.5 µ wide with square ends and
rounded corners.
 Spores : pale to mid green, central or para central in position & do not
swell the sporangium.
 Lipid globules: Black
 Vegetative cytoplasm: Red
6.6 Biochemical tests for confirmation
of B.cereus
6.6.1 Catalase : Positive
6.6.2 Reduction of Nitrate : Positive
6.6.3 Voges-Proskauer (VP) test : Positive
6.6.4 Hydrolysis of tyrosine : Positive
6.6.5 Growth in 0.001% w/v lysozyme : Positive
6.6.6 Liquefaction of gelatin : Positive
6.6.7 Acid production from glucose, mannitol,
xylose & arabinose (in ammonium salt sugar
medium)
6.7 Differentiation of typical strains of
B.cereus from B.thuringiensis
Absence of protein crystals help to
differentiate B.cereus from B.thuringiensis
 B.thuringiensis usually produce protein toxin
crystals in nutrient agar that can be detected
by staining with 0.5% carbol fuchsin.
 Serotyping by H specific antisera
 Molecular Techniques
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