BACTERIAL GROWTH CURVES

&
MEASUREMENT OF GROWTH

BY

Dr. S.V.N.VIJAYENDRA,
Principal Scientist,
FOOD MICROBIOLOGY DEPT.,
CSIR-CFTRI, MYSORE- 570020
INTRODUCTION

GROWTH CURVES
VARIOUS PHASES OF GROWTH CURVE
DIAUXIC GROWTH
FACTORS INFLUENCING THE GROWTH
CONTINUOUS GROWTH
SYNCHRONUS GROWTH

MEASUREMENT OF MICROBIAL GROWTH

A) DIRECT METHODS
B) INDIRECT METHODS
 INTRODUCTION
Orderly increase in quantity of cellular components.
Increase in mass may not reflect growth .
Growth is followed by cell division ( increase in cell number)
Bacterial multiplication is by Binary Fission
Fission increase the cell number in unicellular organisms, whereas,
increases size of the individual in multi cellular organisms

SEQUENCE OF STEPS IN GROWTH:

1) TRANSFER OF NUTRIENTS
2) CONVERSION INTO PROTOPLAST
3) CELL ELONGATION
4) ORGANIZATION OF CONTENTS INTO 2 CELLS
5) INVAGINATION OF THE CYTOPLASMIC MEMBRANE
6) FORMATION OF TRANSVERSE CELL WALL &
SEPARATION OF DAUGHTER CELLS
Binary fission
in
Bacteria
CELL DIVISION IN BACTERIA: BINARY FISSION
1  2  4  8  16  32  64 SO ON….
1  2  22  23  24  2n ;
n = Number of generations
GENERATION TIME:

The time required for the cell/ population to double

Not same for all microorganisms &
Not same for a organism under all conditions

IT DEPENDS ON:
Rate of penetration of medium
Nutrients in the medium
Growth conditions (environment- temp., pH, aeration)
Nature of organisms (genera/ species)
Growth phase etc.
 GENERATION TIME OF DIFFERENT BACTERIA

BACTERIA MEDIUM TEMP. GT

(OC) (MIN)

B. subtilis broth 37 25
B. stearothermophilus broth 60 8
E. coli broth 37 20
E. coli milk 37 12
S. aureus broth 37 30
Lc. lactics milk 37 26
My. tuberculosis broth 37 800-900
Nitrobacter agilis broth 27 1200
Mathematical expression of growth:
Requirements:
No. of cells at time “0”
No. of cells at the end of a given time “T”
Time interval
Total population (N) = 1 x 2n (after T, if a single bacteria is present)
N = N0 x 2n (under practical conditions)
Log10 N = log10 N0 + n log10 2
Log10 N - log10 N0
n= Or n = 3.3 (Log10 N -
log10 N0 )
log 10 2 (or) 0.301

Where n = no. of generations

N0 = no of cells at zero time
N = No. of cells after ‘n’ generations
 Bacterial growth curves

Log no. of cells

No of cells

0 30 60 90 120 150
Time (min)

A) Hypothetical B) Typical growth curve

A TYPICAL BACTERIAL GROWTH CURVE
8
Reasons for Lag phase:
1) The inoculum is in stationary phase or beyond or too old culture
2) When the inoculum is placed in an unsuitable medium or different medium
Theories to explain Lag phase:
1) The organism must modify itself
a) to recover from injury which occurred in the stationary phase
or during the transfer from old medium
b) to adopt itself to the new medium
2) The organism must change the medium by secreting a substance
(ex: enzymes) into the medium, necessary for growth

Xylose
Glucose
Log No. cells

2.5 h lag

Growth of E. coli ( inoculum from exponential growth with arabinose)

 A) LAG PHASE
i) Initial stationary phase (1):
 No increase in cell number, phase of cell enlargement, cells
are active & synthesizing new protoplasm, increase in
total protein , RNA,
 Phase of adjustment necessary for the synthesis of
intermediate metabolites, enzymes, coenzymes.
 Duration varies with species/conditions/media/size of inoculum.
 Prolonged with dormant cells/ phase of culture
 Factors of critical importance for initiation of growth :
pH, Temp., O2 conc. (low / high)

ii) Phase of accelerated growth (2):

 Cell division initiated. Gradual increase in number of cells.
 Rate of multiplication increases with time
 Cells are sensitive to unfavourable environments (extremes of temp.,
osmotic pressure, disinfectants etc).
 It is a transition period.
B) LOGARITHMIC GROWTH PHASE
i) Log growth phase (3)
 Cell division at a constant rate
 Plotting of log of cell number Vs time results in a “straight line”
 Cells in a state of balanced growth. Small in size
 Entire population is uniform in activity.
 Several hours ( species variation, conditions)
Growth rate is maximum & constant
Generation time is shortest & constant
 Use of log phase cultures in fermentation
ii) Phase of decreasing growth rate (4)
 Cells continue to multiply at a slower rate.
(reasons: depletion of nutrients, accumulation of toxic wastes )

C) STATIONARY PHASE (5)

 Cell population remains constant
 Balance between cell division & cell death
 DIAUXIC GROWTH
Growth in 2 separate phases due to the preferential use of
one carbon over the other

Growth of E. coli in equal quantities of glucose & xylose

Highlights : Two exponential growth phases & one transient lag

Reason : Due to catabolic repression of induced enzyme synthesis.
Effect of conc. of carbon source on the growth of Strep. faecalis
(1% yeast extract & 1% peptone)
Hours 0.1% glucose 1% glucose
Cells/ ml pH Cells /ml pH

0 50 x 103 7.4 50x 103 7.4

6 17.5 x 106 6.9 26.8 x 106 5.6
12 32 x 106 6.8 51 x 106 5.1
24 60.9 x 106 6.6 22.1 x 106 4.8

Low temp:

Slow growth, slow deterioration

(longer stationary phase & survival)

Low Oxygen:

Extension of stationary phase

(slow metabolism)
Source: Casciato et al 1975 Appl Microbiol , 29:610-614
D) LOGARITHMIC DEATH PHASE
i) Phase of increasing death rate (6) :
 Decrease in no. of viable cells with increase in time.
 Death rate increases , maximum at the end.
 Transitionary phase

ii) Logarithmic death phase (7) :

 Number of cells decrease exponentially.
(1 Million ½ Million  ¼ Million ⅛ Million………)
 Rate of death constant
 Dying rates are different for different microorganisms
 Steepness & duration of this phase depends on nature & conc. of
toxic wastes (Neisseria is susceptible to autolysis).
E) PHASE OF DECREASING DEATH RATE (8)
Rate of death ≈ Rate of growth
Survival depends on type of microorganism
(Gram negative cocci very rapid ( <72 h), Gram
positive longer periods
Spore formers months & years.)
Biomass

Shivakumar & Vijayendra (2006)

Letters in Applied Microbiol., 42: 477-482
Growth Dynamics in Fed-batch cultivation of Microbial cells
Bacterial growth dynamics in presence of Antibiotic- Chloramphinicol

E. coli growth curve

Source: Ray, 2006

Bacterial Death Curves
Factors affecting growth phases:
 Sudden change in temperature (Room temperature  Ice water)
Curve ceases its upward trend , remain flat for some time & begins to
decline.
 Incubation at < optimum temperature
Rise in +ve phases is much less abrupt & much more extended.

Factors affecting growth (Metabolism & Multiplication) –

Nutrients, water activity, pH, inhibitors, oxygen, light, temperature etc.
Nutrients : (carbon & Nitrogen sources, growth factors (vit. &
minerals),
enzymes, ca, Cobalt, Cu, Fe, Mg, Mn, Molybdinum, K, Zinc)
Water : An essential requirement ( as a solvent, transport of
nutrients & waste, hydrolysis of proteins, fats,
polysaccharides)

EFFECT OF pH:
Affects enzyme activity
Change in permeability of the cell
EFFECT OF TEMPERATURE:
Group Growth temperature
Min Opt. Maximum

Psychrophilic -5 – 0 5-15 15-20

Mesophilic 10-20 20-40 40-45
Thermophilic 25-45 45-60 60-80

Optimum temperature
No. of generations

Maximum growth
temperature

Incubation temperature
 Water activity (aw)
Ratio of the vapour pressure of
water above a material &
the vapour pressure of pure
water.
An index of the available water.
Minimum aw values for
various microorganisms

S. aureus – 0.84-
0.99
Bacillus - 0.90-
0.98
Salmonella – 0.93-0.96
E. coli – 0.94-
0.97
Pseudomonas – 0.96-0.98
Lactobacilli - 0.90-0.94
Penicillium - 0.80-0.90
 CONTINUOUS CULTURE OF MICROORGANISMS
Basis:
 Not all cells are in identical physiological state
(young, actively growing, old & dead cells)
 Effect of chemical substances & physical conditions vary with the phase of
growth
 Maintaining cells in exponential (log) phase essential for optimum
performance.
Solution: CONTINUOUS CULTURE (steady state growth)
2 types: (a) Chemostat, (b) Turbidostat
A) Chemostat:
• Culture volume & cell concentration are at constant
• Conc. of an essential nutrient will control the growth rate of cells
• Conc. is controlled by dilution rate (ratio of amount of the medium flowing

Amount of growth
3Y
in per hr to the volume of culture)
2Y
Growth rate is adjusted by dilution rate
1Y
Low dilution rate: High cell density
Slow growth rate
Low substrate concentration 1X 2X 3X 4X 5X
High dilution rate: Low cell density Amount of limiting nutrient
If dilution rate is low:
• The cells reach a high density, because they
are leaving the culture vessel at a very slow
rate.
• Have time to use the substrate almost
completely, so the substrate conc. is
maintained at a low level.
• This low level of substrate permits the cells to
grow at a slow rate.
If dilution rate is high:
• The cell density is low because the cells are
leaving the vessel a high rate.
• Have little time to utilize the substrate that is
entering the vessel.
• Substrate conc. Is maintained at a high level.
• This high conc. allows the cells to grow at a
Chemostat
high rate.
Maximum stability at low dilution rate
 Turbidostat
B) Turbidostat:

Medium concentration is controlled

by optical sensor
Turbidity of medium is read by absorbance.
Maximum sensitivity & stability at high
dilution rate.

Advantages of continuous culture:

• Constant source of cells in log phase of growth for study of physiology
/genetics

• To study the catabolism of the limiting nutrient.

 SYNCHRONUS CULTURES
A kind of growth in which all cells divide at the same time.
All cells are in the same stage of growth cycle (growing synchronously) for a
few generations
Help to study growth behavoiour (organization, differentiation,
macromolecular synthesis)

How to achieve?

Manipulation of physical environments

Changing composition of medium
Using membrane filters
 EX: 1) Selection by induction method 3) Selection by size & age
(Changing temperature)
B. megaterium (34°C) (6.5 x 106)

Chill 12°C/ 30 min (13 x 10 6)

(shock)

Rapidly increase to 34°C (26 x 106)

(At 12°C cells metabolize but do not divide)

2) Selection by auxotrophic method

Thymine ( DNA constituent)
Essential for cell division

Growth in Thymine free medium

(cells growth but not divide)

Add thymine
Incorporates “T” & DNA divides leading HELMSTETTER –CUMMINGS
to cell division (Synchronous culture) FILTER PAD TECHNIQUE
MEASUREMENT OF MICROBIAL GROWTH
 MEASUREMENT OF MICROBIAL GROWTH

1) Cell counts : a) Indirectly (colony count); b) Directly (microscopy)

2) Cell mass : a) weighing / N2 content ; b) Turbidity
3) Cell activity : Indirectly (biochemical activity)

CATEGORIES OF ESTIMATION METHODS :

1) METHODS BASED ON ESTIMATING COLONY FROMING UNITS

2) MICROSCOPIC OR DIRECT METHODS FOR ESTIMATING

M.O.S

3) METHODS OF ESTIMATING BASED ON MICROBIAL ACTIVITY

4) METOHDS BASED ON IMMUNOLOGICAL RESPONSE

 METHODS BASED ON COLONY FORMING UNITS
1) Plate count method:
“One organism one colony”
Each organism grows, reproducing itself until a visible mass “colony”
appears/develops.
Serial dilutions, plating, incubation at selected temperature, counting etc.
Counting the plates with CFU : 25-250 / plate
Advantage: only viable cells can grow.
Drawbacks: Laborious, variable results (sampling, dilutions, plating,
counting), aerobic / anaerobic, stress due to processing,
differences in nutritional requirements etc.

Modifications:

Plate loop method (calibrated loops)

100 samples/ h (or ) 300-600 / h –automation
Automated dilutors
Spiral palter (50 samples/ h)
Types of
Plating
methods
 SPIRAL PLATE METHOD :

Known volume of sample is dispensed onto a rotating agar plate

in an Archimedes spiral
Amount of sample decreases on the edges.
Counting grid / software to count colonies.
B
Colony count: Colonies in area A +B
Sample volume (in ml) A
Ex: 44+63 = 5.9 x 104 /ml
0.0018
Drawbacks in SPC:
 Failure to form visible colonies on agar medium (nutritionally deficient
unfavourable conditions, cell injury etc)
 Inhibitory substances on glass ware, diluents or produced by competitive
MOs
 Improper sterilization or lack of protection of sterilized diluents, media etc.
 Inaccurate measurement of sample or dilutions mixing or distribution of
sample in diluents
 Improper evaluation of spreaders or pinpoint colonies.

Newer Techniques:
HGMF (Hydrophobic grid membrane filtration)
Petrifilm
Redigel
Simplate
(avoids labour, dilutions, plating)
MEMBRANE FILTRATION METHOD

Large sample volume

Removal of inhibitory compounds by washing the
membrane
Very low numbers of mos can be counted
Samples (water, air, liquid samples)
Any organism can be detected
Rapid method

MPN (most Probable Number ) method:

For samples with very low counts ( ex: water)
MEMBRANE FILTRATION METHOD
Large sample volume
Removal of inhibitory compounds by washing
the membrane
Very low numbers of MOs can be counted
Samples (water, air, liquid samples)
Any organism can be detected
Rapid method
 HGMF: Removal of inhibitors (AOAC* approved method)
Sample Mixing Prefiltration (> 5 micron)

HGMF

Over to Agar plate Reading

-Y&M: YM 11 Agar 48 h at 25°C.
- Salmonella : 24 h.
- Overnight non selective incubation selective broth (6h)

HGMF
(EF 18 Agar) Green colonies
E. coli / coliforms : < 24 h
LMG (Lactose-Manensin-Glucuronate) agar Overnight
Blue colonies
35°C

BMA agar 2 h at 35°C

* Association of Official
Analytical Chemists E. coli (Blue white fluorescent colonies)
Advantages with HGMF methods:
Filtration removes water soluble growth inhibitors & other
undesirable substances (sugars, preservatives etc)
Concentrates MOs – better detection limits
As MFs can be transferred , resuscitation is possible,
before growing on selective media.
As MOs adhere to MFs, they can be stained with specific staining.
(Abs)
PETRIFILM: Film embedded with nutrients are used instead of
agar plates.
One ml of suitable dilution is plated.
Presumptive coliforms

6 h & confirmation – 2 h.

Fecal coliforms (APHA) :

M-7h FC agar

7h 41.5°C

Yellow Fecal coliforms

REDIGEL: Sterile media + Pectin gel in tubes
1 ml liquid sample
Mix Calcium coated plates (ca. pectate gel)

SIMPLATE: Based on MPN method. A special device is used.

By using specific substrate results can be had in 24h
(4- methyl umbelliferone)
Easy to use.
No need of making dilutions and using many media tubes

SIMPLATE
Step 1.
SIMPLATE--procedure
Add 100 mL sterile diluent to medium vessel.

Step 2.
Add 1 mL sample to SimPlate.

Step 3.
Add 9 mL medium to SimPlate.

Step 4.
Swirl SimPlate and pour off excess.

Step 5.
Invert and incubate for 48 hours.

Step 6.
Read results under UV light.
Benefits of SIMPLATE:

Easy:
No agar preparation. Easy to read.
738/plate Most Probable Number (MPN) counting range minimizes
need for dilutions
Rapid:
Instant media preparation. Less than 2 minutes hands-on time.
Reduced read time.

Accurate:
Correlates with US EPA-approved pour plate method using
plate count agar and 48-hour, 35°C incubation.
Simplified procedure minimizes chance of contamination.
Less subjective interpretation than pour plate.

Economical:
Less equipment to purchase and maintain.
Increased productivity, saves labor costs.
Minimal Quality Control (QC)
MPN (most Probable Number ) method:
For samples with very low counts ( ex: water, soft drinks etc.,)
Most Probable Number Calculations
(based on 5 tubes/dilution)
dilutions (100 = 1 ml culture)
100 10-1 10-2 10-3 10-4 MPN/ml
5 5 1 0 0 33
4 5 1 0 0 33
5 4 4 1 0 40
5 4 4 0 1 40
5 5 5 5 2 5400
0 0 1 0 0 0.20
4 4 1 1 0 4.7
Methods based on Microscopy / Direct Detection
Microscopy: Rapid identification (morphology) & counting,
easy to perform,
except microscope no other equip. is required
Breeds method: Direct Microscopic Count ( DMC) 10-15 min/ sample.

Circular marking
Glass slide
Advantages:
Little work is required
Test is not too difficult
Except microscope, no other major equipment is required
Morphology of organisms can be known
Preservatives can be added to extend the holding period
to avoid multiplication of MOs
Only small amount of sample is needed.
Drawbacks: Poor sensitivity, specificity, Viability ? , Laborious,
fatigue, tedious
Rapidity: Membrane filtration / Molecular probes / special staining
 Direct Microscopic Count

In direct microscopic counting:

1) dead cells are not distinguished from living cells
2) small cells are difficult to see under the microscope
3) precision is difficult to achieve
4) we need a phase contrast microscope
5) not a good method for cell suspensions of low density.
Electronic counters (Coulter counter)
Thousands of cells in few seconds
Should be particle free
Rapid & simple
Drawback: viability?
DEFT : Direct Epiflurescent Filter Technique. (On-line)
Milk Pretreatment Filtration (Polycarbonate
membrane)
Staining (Acridine Orange)

Orange –Red (Live) Green (Dead)

Ab-DEFT: E. coli O 157:H7 16 cells / g beef
Listeria : 1h , otherwise 2 weeks
Automation: Bactoscan® - 80 samples / h
Biosensors: On-line method, developments in Optics,
electronics
Rapid, sensitive, versatile, real time analysis
Probes for both Microbes & Toxins.
Probes: Monoclonal or polyclonal antibodies.
Chemiluminescent Biosensor – 10 4 cells / ml
Slide
35
BACTOSCAN
 B) Determining cell mass

DIRECT METHOD:

(a) Fermented broth (b) Cell pellet

Centrifugation protein extraction

Cell mass

Washing
Determining N2 content
Drying the pellet (  14% N on dry weight basis)
Biomass (g/l)

Drawbacks: viability ?,
presence of storage granules (75%) – wrong estimates
 INDIRECT METHOD
Determination of Turbidity (Turbidity- widely used method)
Increase in cell density is directly related to cell number,
At exponential growth, increase in cell mass is directly related to cell number
Rapid & accurate method if standard curve is made

Drawbacks: very high or very low density suspensions

Deeply coloured or with suspended matter
Cannot differentiate dead or living cells.
C) Methods based on Cell Activity: (Indirect Methods)
Degree of biochemical activity ~ Size of population
Dye
A) / nitrate reduction
Impedance
B) method
ATP
C) content
LAL
D) test
Dye
A) reduction : Methylene Blue & Resazurin
Based on the ability of flavine enzymes (dehydrogenases) to
transfer H2 from a substrate to biological acceptor. Dye is reduced.

A H2 MB (Blue) H2 O 2

A MB H2 O2

MB test – 10 5 cfu / 6 h – 37° C

~
 Rate of reduction depends on enzyme activity
 Addition of dye to sample – incubation- reading
 Activity is proportional to bacterial load
 Rapidity : 50-75 samples / h/ person
 Acid forming bacteria have higher reductase activity

Resazurin (blue) Resorufin (pink) [Lovibond comparator]

Advantages of Dye reduction tests:
Cheaper & rapid, natural environment (in milk samples)
•

• No errors even with clumps

• Activity of MOs rather than numbers

Disadvantages with dye tests:


Rate of reduction varies with species, cannot detect type of bacteria

Thermodurics less active at 37°C.

Interference of high somatic cells ( in milk)

Presence of inhibitory compounds reduces the rate

Not much use full with < 105 / ml count
Nitrate reduction test:
Reduction of Nitrate to Nitrite
10 ml sample + 1 ml KNO3 (0.3%) Incubation at 37°C
1 Drop of the mix + 1 drop of reagent* Pink/ Red +ve result
* [5 g Sulphanilamide, 100 ml Glacial acetic acid
1 g alpha Naphtylamine in 100 ml Distilled water]
Ex: Coliforms, Psychrotrophs
Preservatives & antibiotics – inhibit the test
3-5 h [ No colour – pink- red ]
Pre incubation at 15°C , if the period is more than 5 h
75 samples / person. No equipment is required
With a cell count of 2 lakhs --------- 5 H
Not suitable if count is < 50,000 cells
 LAL test: LPS layer in Gram Negative bacteria

Significance in Pharmaceutical products (Injectables)

Extract of Horse shoe crab ( Limulus amoebocyte lysate)
1-2 h at 37°C. Approx 20,000 cfu = 1ng LPS
Qualitative & Quantitative methods
10 samples with 6 dilutions / hour/ person
ATP Method: ON-Line, test time < 1 hour

Luciferin oxidation Luciferase

Relative light units = ATP ; 1 ATP = 1 Photon of

light

 Depends on efficient separation & removal of intrinsic

ATP (Apyrase)
 Quick monitoring in Sanitation & cleanup
 Detection of coliforms with selective enrichment in
6-7 hours
 Potential application in Food, Water & beverage
industries Luminometer
 Commercial kits available
 Sensitivity: 10–12 to 10 –13 g ATP
(10-12 g = 103 bacteria or 1-2 yeast cells)
Lux genes bacteriophage of Salmonella
(luminiscent genes)
Salmonella Salmonella with Lux genes

Luminometer detection (< 45 min)

Method: 500 l Milk
Interfering Factors
500 l NRS-EDTA-Apyrase
pH- 7.75
Temp: 25°C
150 l L-NRB + 50 l Above Turbidity
Colour
100 l Luci- Luciferase

Result (RLU)
IMPEDANCE Method: Resistance to flow of an alternating current
through a conducting material

ANALYZER (Voltage source) 6- 9 hours

with 105 cells
-  Growth - +
Medium
-  - +
-
-  - +
-  - - +
Electrodes

Electrolyte resistance Capacitance

Changes in electrical impedance in medium

 Impedance depends on : Temperature, media composition, Electrode type,
microorganism, Generation time

Psychrotrophs long detection time

Coliforms, Lactobacilli, Staph., Micrococci short detection time

AUTOMATION: BACTOMETER,
MALTHUS-AT
128-256 samples
simultaneously can be analyzed.

METHODS BASED ON IMMUNOLOGICAL METHODS

Mainly based on Antigen – Antibody reaction (Highly specific)
Immunomagnetic separation

Separation of target cells from the complex using immunomagnetic beads

Impedance depends on : Temperature, media composition, Electrode type,
microorganism, Generation time

Psychrotrophs long detection time

Coliforms, Lactobacilli, Staph., Micrococci short detection time
AUTOMATION: BACTOMETER, MALTHUS-AT
128-256 samples simultaneously can be analyzed.

METHODS BASED ON IMMUNOLOGICAL METHODS

Mainly based on Antigen – Antibody reaction (Highly specific)
Immunomagnetic separation
Separation of target cells from the complex using immunomagnetic beads
Table- List of commercially available immunomagnetic particles

Particles Size (m) Manufacturer

BioMag 4300 Super paramagnetic 0.5-1.5 Metachem Diag.
UK
Polystyrene paramagnetic microparticles 1-2 Polysciences Ltd,
UK
Magnetic polyacrolein/iron oxide particles 1-10 Scipac, UK
Polystyrene/ Divinyl- benzene 0.7 Sepadyn, USA
Dynabeads M –280/ M-450 2.8 / 4..5 Dynal A/S, Norway

Methods:
1) ELISA : Staph., salmonella, Listeria, E.coli, B. cereus, Toxins
Automated VIDAS®
2) Latex Agglutination : Commercial kits
Ex: Clear view ® - Ab to flagellars AG of Listeria
96 well ELISA Plates
ELISA Plate Reader
Automated identification Systems:

Auto mated identification Systems:

Ex: Vitek; Autoscan, Walk away, Biolog, MIDI
(semi to complete automation)
Vitek; Autoscan, Walk away, Biolog, MIDI
(semi to complete automation)

VIDAS VITEK
 METHODS BASED ON
MOLECULAR BIOLOGICAL TECHNIQUES
Gene Probes :
for detection, identification & characterization of pathogens
Few bases Several thousands

Labeled nucleic Acid probes Complementary Target Sequences

If double strands formed Test is + ve.

Polymerase Chain Reaction (PCR) : A rapid method
Single copy of gene several thousands in few hours
Nested PCR : 2 cfu / ml (Salmonella); Hiochi bacteria : 10 cells / ml
10 cfu pathogens/ 100 ml water with 8700 cfu SPC/ml
3 major steps:
I. Template (DNA) preparation:
Sample Culture isolation
Overnight at 37°C 150 rpm
Centrifugation ( 8500 / 5”)

Washing of pellet (3 times) PBS pH7.4

Wash with st. Distilled water

Suspend in 50 l st. D. water & dilute with 1:10 Triton X-100

Boiling for 5” and cool on ice.

II. PCR:
1 l of dNTPS + 0.6 l of Taq (1.5 U) polymerase + 5 l Tris buffer
4-5 l template Add gene specific primers (0.7-0.9 picomoles)
Make up to 50 l with St. milliq water

PCR M/C (pre set)

(Denaturation, annealing, extension- elongation )
90-95°C 45-60°C 72°C
Repeat the cycles
III. GEL electrophoresis:
Sample after PCR

Electrophoresis (Agarose gel 1.5%) in 1x TAE Buffer [1.5 - 2 hours]

Staining the gel (ethidium bromide )

Observation for DNA bands in UV light & Documentation ( Gel - Doc system)
Table: Chart showing the organisms and their specific target genes
Used in detection by PCR as probes / primers
Bacteria Virulent gene
Aeromonas Enterotoxin
Bacillus cereus Enterotoxin
Campylobacter Flagellar protein (MOMP)
Clostridium plc gene, enterotoxin A
Helicobacter Urease, membrane lipoprotein gene
E.coli Lecithinase
Klebsiella Heat labile enterotoxin
heat stable enterotoxin
Shigella Shiga toxin, vir, ctx gene
Vibrio hly A, ctx gene
Yersinia v- antigen , w- antigen
 RAPID IDENTIFICATION KITS
Conventional method: Preparation of several media
in a single tube
Ready-to-use kits to carry out biochemical tests. Easy to use, no preparation.
Ex: API, Enterotube,
Micro-ID, Rapid one, Spectrum
etc
 RAPID IDENTIFICATION KITS
Conventional method: Preparation of several media in tubes

Ready-to-use kits to carry out biochemical tests

Ex: API, Enterotube,
Micro-ID, Rapid one, Spectrum
etc

Easy to use, no preparation.

With a single step inoculation 10-15 tests can be carried out

 Over night incubation and observe for results
 Get a number and identify the bacteria using database
provided
WHERE AND HOW OF MICROBIOLOGICAL ASSESSMENT
PROCESSING STAGE APPLICATION OF TECNIQUE

RAW MATERIALS, WATER CONVENTIONAL TECH./ RAPID TECH/

NUCLEIC ACID PROBES

READY FOR PACKAGING DISTRIBUTION POINTS RAPID TECH.

BULK STORAGE/
READY FOR PROCESSING CONEVNTIONAL TECH.

PROCESSING LINE ATP, HGMF,IMMUNE DETECTION,

CCP RAPID BIOCHEMICAL TESTS,
MOLECULAR TYPING

PACKAGING & STORAGE CONVENTIONAL TECH./RAPID TESTS

DISTRIBUTION & RETAIL AUTOMATED SYSTEMS, HGMF,

IMAGE ANALYSER

CONSUMER’S REPORT CONVENTIONAL TECH.,

SURVEILLANCE DATABASE MOLECULAR TYPING, ELISA KITS
Features of the ideal automated microbiological assay system:
1) Accuracy ( fort the inteded use purpose) interms of
sensitivity : minimal detectable limits
Specificity: of the system
versatility : Potential applications
2) Speed in productivity : No of samples / run / shift / day
3) Cost:initial, per test, reagents etc.
4) Acceptability : by regulatory or by scientific community
5) Simplicity of operation, sample treatment, operation of equipment,
computer versatility
6) Training: on site, how long, quality of training personnel
7) Reagents: preparation / stability/ availability/ consistency
8) Reputation of the company of the equipment / kit manufacturer
9) Technical service : speed & availability , cost
10) Utility & space requirement
References:

Basic Food Microbiology- Banwart

Microbiology _ Pelczar et al
General microbiology- Stanier et al
A comprehensive dairy microbiology - Yadav et al
Food Biotechnology – Y.H. Hui & George G.K. (VCH
publications)
General Microbiology – Vol-II -Powar CB & Daginawalia HF

Micha Peleg & Maria G. Corradini (2011): Microbial Growth

Curves: What the Models Tell Us and What They Cannot.
Critical Reviews in Food Science and Nutrition, 51:917-945