Applied microbiology encompasses many aspects
of modern microbiology. We use microorganisms to
produce many of the foods we eat such as cheese,
yogurt, bread, sauerkraut, and a whole list of fermented
beverages.
Their presence and numbers in our foods
and drinking water determine if it is safe to consume
these substances as they could cause us harm and
disease.
The presence of microorganisms in food does not necessarily
indicate that the food is spoiled or that it has the
potential to cause disease. Some foods can have high
counts because microorganisms are used in their production.
Yogurt, sauerkraut, and summer sausage are examples of
foods prepared by microbial fermentation and, therefore, they
have high bacterial counts associated with them during
production.
Post- production treatments such as pasteurization or
smoking will significantly reduce the numbers of bacteria
present.
During processing and preparation, food can become
contaminated with bacteria, which naturally occur in the
environment
Milk in the udders of healthy cows is sterile, but bacteria such
as Streptococcus and Lactobacillus are introduced during
milking and processing because they are part of the bacterial
flora associated with the animal, especially the outside of the
udder.
Pasteurization kills many of the bacteria that are introduced
during processing, and any pathogens that may be present,
but it does not kill all the bacteria present in milk. Some
bacteria in milk can survive pasteurization temperatures and
eventually cause spoilage and souring of milk. These are called
thermoduric bacteria.
Food-borne illnesses
We must also bear in mind that food can be an important
means for the transmission of disease.
The Centers for Disease Control estimates that 76 million
people per year in the United States become sick; 300,000 are
hospitalized; and 5,000 people die from foodborne illnesses.
This usually result because pathogenic bacteria or their toxins
are introduced into food products during processing, handling,
or preparation.
Staphylococcus aureus (food poisoning)- Unsanitary practices
by the food handler
Clostridium botulinum (botulism)-Endospores grow in
improperly home-canned foods. The endospores occur in the
soil and the environment and contaminate the prepared
vegetables.
Salmonella and Campylobacter
and eggs
associated with poultry
Escherichia coli O157:H7 Associated with meat if fecal
material from the animal’s intestines contaminates meat
during the butchering process.
Common Food Borne Pathogens
Coliform Bacteria
Although high bacterial counts in food do not necessarily
mean that the food is spoiled or that it harbors diseasecausing organisms, it can suggest the potential for more rapid
spoilage of the food.
One method to ascertain if food is contaminated with fecal
bacteria and, therefore, has the potential to spread disease is
to perform coliform counts.
Coliforms are organisms such Escherichia coli that occur in the
intestines of humans and warm-blooded animals.
Their presence in food or water indicates that fecal
contamination has occurred and that there is the high
potential for the spread of serious disease such as typhoid
fever, bacillary dysentery, cholera, and intestinal viral
diseases.
Coliform Counting
The standard plate count and the coliform count can be used
to evaluate foods in much the same manner that they are
used on milk and water to determine total bacterial counts
and the number of coliforms.
For solid food: food suspension should be made (via food
blender) -BEFORE COUNTS ARE PERFORMED!
Conditions affecting bacterial growth:
•
FOOD
-bacteria need food for reproduction
-HIGH PROTEIN FOOD and STARCH FOOD promote bacterial
growth!
•
ACIDITY
-most bacteria require specific pH range in order for them to
carry out processes
pH ranges of bacteria
4. TEMPERATURE
--bacterial proliferation can be controlled by heat and cold
-use of refrigeration temperatures slows down bacterial
proliferation. The colder the better.
PASTEURIZATION
-refers to cooking of food where only a certain number of
bacteria are killed. But it does not render the product sterile.
STERILIZATION
-total destruction of living organisms. Only used for canned
shelf-stable items
 Clostridium botulinum: resistant to heat . They
produce toxins that causes paralysis and are fatal.
Grow best at 60-115F. Cool all food fast after heating!
 Canning procedure: cook in a retort or pressure
device of 240 F-250F for several hours
5. OXYGEN
-AEROBIC: bacteria that needs oxygen for growth
-ANAEROBIC: bacteria that does not require oxygen for growth
-TAKE NOTE: Most bacteria that responsible for food spoilage
REQUIRE OXYGEN; Most pathogenic bacteria are anaerobic
6. MOISTURE
-amount of water available in food for chemical reactions and
microbial growth is called Water Activity (Aw)
-water activity is measured from 0 (totally dry) to 1.0 (pure
water)
-most bacteria can grown only in food that have water activity
higher than 0.90
-the water activity available in food can be reduced by
freezing, dehydration, adding salt or sugar or other water
binders.
Water Activity
Types of Bacteria on food: Spoilage
Food preservation
Controlling Bacteria : History
Most of the old methods of food preservation were:
-cavemen discovering fire to cook meat
-use of salt by Egyptians
-sausage drying and fermentation by the Europeans
Controlling bacteria: Controlling bacteria counts
The single most important control factor: KEEPING BACTERIAL
COUNTS ON RAW FOOD AS LOW AS POSSIBLE
Action:
● Keeping raw material as clean as possible
● proper cleaning of food processing facilities
● Use of sanitizing solutions such as chlorine in
sanitizing plants
● Proper instruction for food handlers and staff
designated in food processing and make them aware
of proper hygiene practices
Controlling bacteria: Temperature
 Bacterial proliferation can be controlled by heat and
cold.
 Bacterial generally do not grow at freezing
temperatures but they can survive.
Use of refrigeration temperatures (30-45 F): slows down the
rate of bacterial proliferation.
THE COLDER, THE BETTER.
Pasteurization of food= reduction of bacterial population for
up to 90-95%
Milk is a good example. Milk is heated to a high temperature
(161 F) for a very short period of time (15 seconds) but it will
still spoil due to unkilled bacteria after extended refrigerated
storage.
Fun fact: Milk pasteurization started in the early 1950s mainly
to reduce the incidence of Mycobacterium tuberculosis.
Controlling bacteria: Control oxygen
Bacteria that cause spoilage are mostly AEROBIC.
By eliminating the oxygen in food: drastic slow of bacterial
proliferation.
Meat are now sold in vacuum seal bags.
Controlling bacteria: Control moisture and acidity
Control of water of by determining the water activity is the
key!!
In high sugar items like honey jellies, and molasses do not
generally spoil from bacteria because the sugar ties up the
available moisture.
Low pH= control and prevent bacterial growth!
Water Bacteriology
Prior to the modern age of public health, water was a major
means for the spread of infectious diseases such as cholera,
dysentery, and typhoid fever. A physician, John Snow, showed
in the 1840s that a cholera epidemic in London was the result
of cesspool overflow into the Thames River from a tenement
where cholera patients lived.
When water for drinking was drawn by inhabitants near the
cesspool discharge, the contaminated water and pump
became the source for the spread of the disease to people in
the area. Snow’s solution was simply to remove the handle to
the pump, and the epidemic abated.
From a microbiological standpoint, it is not the numbers of
bacteria that are present in water that is of primary concern to
us but rather the kinds of bacteria.
Water found in rivers, lakes, and streams can contain a variety
of bacteria that may only be harmless saprophytes, which do
not cause disease in humans.
However, it is important that water not contain the intestinal
pathogens that cause typhoid, cholera, and dysentery. In
modern cities, treated sewage is discharged into receiving
waters of lakes, rivers, and streams, and this constitutes a
major sanitary problem because those same bodies of water
are the sources of our drinking water.
As a result, we have developed methods to treat water to
eliminate the potential for disease, and we do microbiological
tests to determine if water is potable and safe for
consumption.
At first glance, it might seem reasonable to directly examine
water for the presence of the pathogens Vibrio cholerae,
Salmonella typhi, and Shigella dysenteriae. However, this is
not the case because it would be tedious and difficult to
specifically test for each of the pathogens.
Furthermore, these bacteria are often fastidious, and they
might be overgrown by other bacteria in the water if we tried
to culture and test for them. It is much easier to demonstrate
the presence of some indicator bacterium, such as Escherichia
coli, which is routinely found in the human intestine but is not
found in the soil or water.
The presence of these bacteria in water would then indicate
the likelihood of fecal contamination and the potential for
serious disease.
Indicator of water contamination:
E. coli is a good indicator of fecal contamination and a good
test organism.
This is for several reasons:
(1) it occurs primarily in the intestines of humans and some
warm-blooded animals and it is not found routinely in soil or
water;
(2) the organism can be easily identified by microbiological
tests;
(3) it is not as fastidious as the other intestinal pathogens, and
hence it survives a little longer in water samples.
Examples of Waterborne pathogens :
Coliform bacteria
By definition, organisms such as E. coli and Enterobacter
aerogenes are designated as coliforms, which are gramnegative, facultative anaerobic, non-endospore-forming rods
that ferment lactose to produce acid and gas in 48 hours at
35C.
Lactose fermentation with the formation of acid and gas
provides the basis for determining the total coliform count of
water samples in the United States and therefore
designateswater purity.
The presence of other bacteria, such as Enterococcus faecalis,
which is a gram-positive coccus that inhabits the human
intestine, can also indicate fecal contamination, but testing for
this bacterium is not routinely done in the United States
Laboratory tests:
•
Plate count
-counting no. colonies in pour plate cultures
-on Nutrient agar plates at 37 degrees Celsius for 1-2 days
And at 22 degrees Celcius for 3 days.
Growth at:
22: mainly saprophytic; indication of a decomposing matter in
water
37: most likely of human origin; index of dangerous pollution
Nutrient agar plate
2. Coliform Count: Presumptive Test
● It is used for the detection and estimation of coliform
in the water sample.
● For estimation of coliforms: Lactose containing Broth
Medium is used.
● Commonly used medium: Mac Conkey Broth with
Bromocresol Purple
● An inverted Durnham’s tube is placed
● Color of the media changes into yellow and on
collection of gas in the Durnham’s tube: POSITIVE (A
coliform is present)
● The number of positive tubes are counted and
referred to the standard chart to find MPN of total of
100 ml water sample.
Mac Conkey Broth with Bromcresol Purple Indicator and
Durnham’s tube
Coliform Count: Presumptive Test
In the presumptive test, 15 tubes of lactose broth are
inoculated with measured amounts of water to see if the
water contains any lactose-fermenting bacteria that produce
gas.
If, after incubation, gas is seen in any of the lactose broths, it
is presumed that coliformsare present in the water sample!!
This test is also used to determine the most probable number
(MPN) of coliforms present per 100 ml of water.
This 15-tube procedure is most applicable to clear surface
waters but can be used for brackish water and waters with
sediments.
Coliform Count: Confirmed Test
In this test, plates of Levine EMB agar or Endo agar are
inoculated from positive (gas-producing) tubes to see if the
organisms that are producing the gas are gram-negative
(another coliform characteristic).
Both of these media inhibit the growth, of gram-positive
bacteria and cause colonies of coliforms to be distinguishable
from non-coliforms.
On EMB agar: coliforms produce small colonies with dark
centers (nucleated colonies).
On Endo agar: coliforms produce reddish colonies. The
presence of coliform-like colonies confirms the presence of a
lactose-fermenting, gram-negative bacterium.
Coliform Count: Confirmed Test
Once it has been established that gas-producing lactose
fermenters are present in the water, it is presumed to be
unsafe.
However, gas formation may be due to noncoliform bacteria.
To confirm the presence of gram-negative lactose fermenters,
the next step is to inoculate media such as Levine eosinmethylene blue (EMB) agar or Endo agar from positive
presumptive tubes.
Coliform Count: Confirmed Test
Levine EMB agar contains methylene blue, which inhibits
gram-positive bacteria. Gram-negative lactose fermenters
(coliforms) that grow on this medium will produce “nucleated
colonies” (dark centers).
Colonies of E. coli and E. aerogenes can be differentiated on
the basis of size and the presence of a greenish metallic sheen
E. coli colonies on this medium are small and have this
metallic sheen, whereas E. aerogenes colonies usually lack the
sheen and are larger.
It should be remembered that E. coli is the more reliable
sewage indicator since it is not normally present in soil, while
E. aerogenes has been isolated from soil and grains.
Endo agar contains a fuchsin sulfite indicator that makes
identification of lactose fermenters relatively easy.
Coliform colonies and the surrounding medium appear red on
Endo agar.
Nonfermenters of lactose, on the other hand, are colorless
and do not affect the color of the medium.
In addition to these two media, there are several other media
that can be used for the confirmed test:
•
Brilliant green bile lactose broth,
•
Eijkman’s medium,
•
EC medium
Brilliant Green Lactose bile Broth with Durnham’s Tube
Eijkman’s Medium with Durnham’s tube
Coliform count: Completed Test
In the completed test, our concern is to determine if the
isolate from the agar plates truly matches our definition of a
coliform. The media for this test include a nutrient agar slant
and a Durham tube of lactose broth.
If gas is produced in the lactose tube and a slide from the agar
slant reveals that we have a gram- negative, non-sporeforming rod, we can be certain that we have a coliform
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and Q fever. •
Diphtheria
Cow pox,
HFMD
Leptopirosis,
Campylocabter,
Anthrax
METHODS FOR DISINFECTION/STERILIZATION OF MILK
•
A final check of the colonies that appear on the confirmatory
media is made by inoculating a nutrient agar slant and lactose
broth with a Durham tube.
After incubation for 24 hours at 35C, the lactose broth is
examined for gas production.
A Gram-stained slide is made from the slant, and the slide is
examined under oil immersion optics.
If the organism proves to be a gram-negative, nonsporeforming rod that ferments lactose, we know that coliforms
were present in the tested water sample
Nutrient agar slant
Lactose Broth with Durnham’s tube
The completion of these three tests with positive results
establishes that coliforms are present; however, there is no
certainty that E. coli is the coliform present. The organism
might be E. aerogenes.
Of the two, E. coli is the better sewage indicator since E.
aerogenes can be of non-sewage origin. To differentiate these
two species, one must perform the IMViC tests,
3. Membrane Filtration Method
-Measure quantity of water filtered through a millipore filter.
-bacteria will be retained on the surface
-filter paper placed on media face upwards and then
incubated
-colonies to be counted
-presumtive coliform and E. coli count after 18 hours of
incubation
Bacteria Examination of Water
-
Should be done regularly, periodical procedure
Drinking water: free of pathogenic organisms
Classification of drinking water
Milk Bacteriology
Types of bacteria in milk
Milkborne diseases
● Mostly TB
● Brucellosis
● Coxiella
● Streptococcus Infections
● and Staph food poisoning,
● Salmonellosis
•
•
THERMIZED MILK: 57-68 deg C for 15 sec (Methylene
blue test)
PASTEURIZATION: HOLDER METHOD (63 deg C for 30
min)/ FLASH (72 deg C for 20 sec) hence does NOT
effectively kill: Bacterial pores, Coxiella,
Mycobacterium, some toxins
UHT: 135 deg C for 1 sec (Viable count test: ≤
1000/mL aftr incubating on yeast extract medium for
48 hrs)
•
4. STERILIZATION: 100 deg for extended period to
pass TURBIDITY TEST
Bacteriological examination of milk
•
VIABLE COUNT:
•Serial Plate dilution in YE milk agar
•Incub atr 30 or 21 deg c for 72 hrs
•Number of colonies multiplied by dilution factor give
COLONY COUNT
•
COLIFORM COUNT:
•Serial dilution of milk inoculated in 3 tt MacConkey broth
with Durham’s tube
•Incub. at 37 deg C for 48 hrs and acid+gas looked for
Bacteriological examination of milk
Bacteriological examination of milk
Examination of specific pathogen
• Tubercle bacillus
• Milk 3000rpm x 30 mins.
• Sediment --- LJ medium/2 guinea pigs.
• Observe animals for 3 months.
• Even culture can also be done
Examination of specific pathogen
2. Brucella
• Inoculate cream on serum dextrose agar / centrifuged
deposit i.m. in guinea pigs • Animals:
- ANTIBODIES: Milk ring test/ whey agglutination test
- sacrificed after 6 weeks and tested for antibodies. Spleen
inoculated in culture media
Examination of specific pathogen
3. Clostridium .perfringens:
• Incubate different qty of milk in litmus milk medium
(anaerobically) at 37deg C x 5days and look for stormy
fermentation
Thank you.
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