Food Microbiology &
Helen Billman-Jacobe
Control of microorganisms in food
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Ray and Bhunia Ch 33 Control by heat (thermal processing
Intended learning outcomes
Define bacteriostatic, bacteriocidal and bacteriolytic
Know the purpose of pasteurization and decimal reduction time
Describe the difference effects of low and high thermal processing methods Express D, TDT and z values
Kill or inhibit growth
Bacteriostatic = can inhibit growth of bacteria without killing them Bactericidal = can kill bacteria
Bacteriolytic = lyses (breaks open) bacterial cells
Viable counts
Serial decimal dilution
Spread plate method
Known volume of diluted sample spread on plates Count the colonies
Calculate cfu/ml
Only viable, culturable cells will form colonies Colonies/(volume plated ml)X dilution factor) = cfu/ml
Direct counts
If cells can be viewed under a microscope then they can be counted using a special microscope slide, counting chamber = hemocytometer
1. Clean the chamber and cover slip with alcohol. Dry and fix the coverslip in position.
2. Harvest the cells. Add 10 μL of the cells to the hemocytometer. Place the chamber in microscope
3. Count the cells in the gridded square
4. Multiply by the conversion factor to estimate the number of cells per mL.
5. Prepare duplicate samples and average the count.
Neubauer chamber’s counting grid
is 3 mm x 3 mm in size.
The grid has 9 square subdivisions of width 1mm
In case of blood cell counting, the squares placed at the corners are used for white cell counting. Since their concentration is lower than red blood cells a larger area is required to perform the cell count.
The central square is used for platelets or red cells . This square is split in 25 squares of width 0.2 mm (200 μm)
Each one of the 25 central squares is subdivided in 16 small squares. Therefore, the central square is made of 400 small
squares and can be used to count yeast of bacteria
Counting cells
Look for the first counting grid square where the cell count will start.
This example is from a Neubauer-improved chamber
Start counting the cells in the first square then move to the next
Different laboratories have different counting protocols, however, there is a popular unwritten rule that states:
“Cells touching the upper and left limits should be counted, unlike cells touching the lower and right limits, which should not be taken into account.”
Calculation
Concentration = Number of cells x 10,000 / Number of squares
We apply the formula for the calculation of the concentration:
Concentration (cell / ml) = Number of cells / Volume (in ml)
The number of cells will be the sum of all the counted cells in all squares counted.
Since the volume of 1 big square is:
0.1 cm x 0.1 cm = 0.01 cm2 of area counted.
Since the depth of the chamber is 0.1mm:
0.1 mm = 0.01 cm
0.01 cm2 x 0,01 cm = 0.0001 cm2 = 0.0001ml = 0.1 μl
So, for the Neubauer chamber, the formula used when counting in the big squares is:
Limitations and advantages of counting methods
Thermal treatment of food
The basic purpose for the thermal processing of foods is
to reduce or destroy microbial activity reduce or destroy enzyme activity and
to produce physical or chemical changes to make the food meet a certain quality standard.
Summary of thermal processing methods
Low-heat processing or pasteurization:
Foods are heated at temperatures below 100°C for a fixed time with the objectives to kill all pathogens, except spores, and ~ 90% of spoilage microbes, except thermoduric bacteria, spores, and toxins.
High-heat processed foods:
Foods are heated uniformly at 100°C or above for the time, which depends on a product and microbes to be killed. For low acid foods a 12 D treatment is given to destroy C. botulinum spores, and get commercial sterility.
Microwave heating:
When frozen foods are treated with microwaves, the waves change polarity rapidly, making water molecules to move fast. The frictions of the water molecules generate heat, which kills bacteria. However, the food may not be heated uniformly and can have cold spots. (cannot assure complete destruction of pathogens)
Mechanism of thermal inactivation of microbes
Vegetative cells
• injury to cell membrane (more permeable)
• DNA (strand breaks), ribosomal RNA breakdown • Cell wall damage
Mechanism of thermal inactivation of microbes
Bacterial Spores
• lose components of spore coat
• damage to layers that become membrane and
• cannot take up water for germination
Coat Outer membrane Cortex Germ wall Inner membrane Core
Thermal treatment: time
Three common and important questions regarding thermal inactivation of bacteria in foods:
1. How much time (at x°C) is needed to kill bacteria present?
2. How do different bacteria vary in their sensitivity to heat?
3. What happens to kill rate as I increase the temperature?
Three well known values:
D-value, Thermal Death Time, z-value
Decimal reduction time (D-value):
At a given temperature (C or F), the time takes (D min) to kill 90% (1 log) of a microbial population (cells, spores).
D-values can be used to determine relative heat sensitivity among species and strains, as well as, what heating time is required (D values), under a given time- temperature combination, to reduce a microbial population to certain level.
10 15 time minutes
D value formula
DT value = t2 – t1 /(log N0 – log N1)
T = temperature
t1 initial time
t2 = final time
N0 = initial population N1= final population
0 5 10 15 20 25 time minutes
Thermal treatment
Microbial destruction in food by heat is expressed in terms of its exposure to a specific temperature
for a period of time
1.E+11 1.E+09 1.E+07 1.E+05 1.E+03 1.E+01
1.E-01 0 1.E-03 1.E-05 1.E-07 1.E-09
150 200 250 300
Killing curve of C. botulinum: This curve presents the DR value (12.6 seconds) and the 12-D reduction (151 seconds) for C. botulinum. The killing agent is heat at 121°C.
time seconds
12D process
Experience has shown that a process equivalent to twelve decimal reductions in the population of C. botulinum spores is sufficient for safety; this is referred to as a 12D process
Assuming an initial spore load of 1 spore/g of product, it can be shown that, for such a process, the corresponding probability of C. botulinum spore survival is 10-12, or one in a million million.
This implies that for every million million cans given a 12D process, and in which the initial load of C. botulinum spores was l/g, there will be only one can containing a surviving spore.
This is commercially acceptable
Decimal reduction times (D values) for bacterial spores
B. stearothermophilus
C thermosaccharolyticum D. nigrificans
C. botulinum (types A & B) C.sporogenes (PA 3679) B. coagulans
C. botulinum type E
Approximate optimum growth temp. (°C)
D value (min) a/
D121.1 4.0 – 5.0 D121.1 3.0 – 4.0 D121.1 2.0 – 3.0 D121.1 0.1 – 0.23 D121.1 0.1 – 1.5 D121.1 0.01 – 0.07 D82.2 0.3 – 3.0
Thermal death time (TDT)
100,000,000.0 10,000,000.0 1,000,000.0 100,000.0 10,000.0 1,000.0 100.0 10.0 1.0
02004006008001000
-time required for complete destruction of specific number of microbial cells or spores at a specific temperature.
Thermal death time (TDT)
-time required for complete destruction of specific number of microbial cells or spores at a specific temperature.
Higher temperature
Shorter TDT
Slope of line steeper than at lower temp
100,000,000 10,000,000 1,000,000 100,000 10,000 1,000 100 10 1
02004006008001000 seconds
100,000,000.0 10,000,000.0 1,000,000.0 100,000.0 10,000.0 1,000.0 100.0 10.0 1.0 0.1
62°C 65°C 100,000,000
10,000,000 1,000,000 100,000 10,000 1,000 100 10 1
0200400600800100002004006008001000 seconds 0 seconds
Thermal death time (TDT) and Z value
60 62 64 66 68 70 72 74
temp of thermal treatment
The z-value is a measure of the change of the D- value with varying temperature
the slope of the curve gives Z value (time to reduce D-values by 1 log
The D value at 62°C is 139.9 sec and at 71°C it is 12.9.
Ray and Bhunia Ch 33 Control by heat (thermal processing
D values (min, log scale)
Control by heat Pasteurisation
• uses temperatures below 100°C
• aim is to destroy all vegetative cells of pathogens, and a large proportion (90%) of spoilage microbes
• Time/Temp. combination used is designed to be minimum to achieve microbiological standards, and minimize thermal damage to food (quality)
• Thermoduric bacteria survive (e.g. spore-formers)
• Refrigeration normally used to delay spoilage
• Bacterial heat-stable enzymes can be a problem
• Milk is most obvious food item that is pasteurized
Thermal processing of dairy food from FSANZ Standard 4.2.4
(1) Milk must be pasteurised by –
(a) heating to a temperature of no less than 72°C and retaining at such temperature for no less than 15 seconds; or
(b) heating, using any other time and temperature combination of equivalent or greater lethal effect on any pathogenic micro-organisms in the milk
In the case of Ultra Heat Treatment (UHT) of milk, for example, temperatures of at least 132°C must be used to achieve commercial sterility.
Thermal processing of dairy food from FSANZ Standard 4.2.4
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