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DATE :9/10/2016 1
al growth/ control of microbi Microbial growth
The method of counting bacteria is divided into: 1) direct 2) indirect
Viable cell counts: Plate counts: Serial dilutions put on plates CFUs form colonies
Plate count is one of direct methods for counting bacteria
2
There is two types of plate count: 1) the pour plate method 2) the spread
plate method
The difference between them is that I have to dilute the sample whether the
sample is water, juice, milk …etc. . . .
We don’t usually use this method for urine sample.
10^-2, …) Dilution in bacteria is log dilution
(10^-1,
In the growth curve in the previous lecture ‘e used time vs. log number of bacteria
not the time vs. the number of bacteria
So in the first type (the pour plate method) we used *antipetri dish containing 1
ml of *every solution with 13 ml of melted agar and mix it then leave it to
dry for *30 minute and put it upside down in the incubator 3
(2:39) the number of colony used in the plate more than 30 and less than 300 (30-
300)
Some food microbiologists use from 25 to 250
In the second type we have petri dish and take 0.1 ml for each solution* then we
spread the liquid with a spreading glass over the surface of the agar then
leave it to dry and put it in the incubator then we count the number of
bacteria
When we count the colony forming unit = number of colony * dilution factor but
not to the power of minus because we want to return to the original
number of bacteria in the sample
(4:50) the device is a colony counter which makes the plate larger so it is easier for
the technician to count the number of colonies and it is supposed to give us
similar number in any plate we use
Why? Because if the number is small we multiply it by high dilution factor and vice
versa
So in the first type we use one ml and in the second we use 0.1 ml and spread, it
to even it out
We should multiply it with correction factor 10 (number of colonies *10) 4
Some of the disadvantages of this method is that some bacteria is sensitive to
heat and the melted agar temperature is 55C also some bacteria will grow
on the subsurface (beneath the actual surface)
So the obligatory aerobic bacteria cannot survive these conditions
Direct microscopic count: Counting chambers (slides) for microscope
A specially designed slide called a petroff-hausser cell counter
is used in direct microscopic counts.
-motile bacteria are difficult to count by this method
-Dead cells are bout as likely to be counted as live ones
.
This method is also direct and use counting chambers .This method calculate
the total number of bacteria (dead and viable cells)
Viable cells can divide and form colonies unlike dead cells (cannot divide so
they do not form colonies)
Filtration is the method of choice for low counts M.O.
• Membrane filters for fluids.
Pore size for bacteria: 0.2 – 0.4 m
Pore size for viruses: 0.01 m
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At least 100 ml of water are passed through a thin membrane filter whose
pores are too small to allow bacteria to pass.
Filtration is a direct method used with low concentration of M.O
(microorganism)
In this method we use membrane filters which have pores of specific size so
the fluid go through the pores but the bacteria cannot and stays on the
surface of the membrane , it cannot be used with high concentration
because the bacteria is in high concentration it will block the pores
For the bacteria the pore size is (0.22-0.45 micro m in diameter)
We take the filter and put it on petri dish (selective media) and the water will
transport from the media to the membrane filter and after 24
hours we will obtain colonies of bacteria that we can count
If the sample is water and contain heavy metals cannot used this method
because the heavy metals will block the pores
Only viable cells is counted in this method
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Indirect methods
Spectrophotometry to measure turbidity
OD is function of cell number
Spectrophotometry is an indirect method
Spectrophotometry device is used to measure absorbance
In the second test tube most of the light will hit the bacteria and will be
scattered and some of the light will reach the device
Direct relationship between absorbance and the number of bacteria
Indirect relationship between transmission and number of bacteria
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The most probable number (MPN) method
- Another method for determining the number of bacteria in
a sample is the most probable number (MPN) method
- This statistical estimating technique is based on the fact that
the greater the number of bacteria in a sample, the more
dilution is needed to reduce the density to the point at which no
bacteria are left to grow in the tubes in a dilution series.
- It is useful when the growth of bacteria in a liquid differential
medium is used to identify the microbes (such as coliform
bacteria, which selectively ferment lactose to acid, in water
testing).
تعتمد على عامل التخفيف بنعمل تخفيف لمرحلة اعداد البكتيريا قليل جدا
Used mostly for water samples to identify microbes usually coliform bacteria
Coliform bacteria is: 1) E.coli 2)klebsiella 3) enterobacteria
And their presence is an indicator for fecal contamination in the water, the
more the contamination of the sample the more dilution is
needed
We use selective media that depends on trait of coliform bacteria, coliform
bacteria produce acid and gas during fermentation, the acid
will change the color of the media and the gas will be
collected in small tubes called Durham tubes
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We take three sets of test tube each set composed of five test tube that
contain 10 ml of selective media
The empty spaces in the tubes are gas
4 positive result ( contain gas )
10 ml of ‘water sample
First test
3 positive result
1 ml of ‘water sample
Second test
1 positive result
0.1 ml of ‘water sample
Third test
The whole water samples should be 100 ml
After incubation will see change in color and gas formation
We take the positive result (in our case 4-3-1 ) and compare it with the
standard table (slide 33)
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The WHO divide water purity to 4 groups : <2 pure water, 2-8
acceptable
8-40 doubtful, >40 swage water
Q: will the bacteria grow in the time needed to make the tests?
No because microbiology departments always have these test ready all they
have to do is obtain the samples
Metabolic activity
- indirect way to estimate bacterial numbers is to measure
A population's metabolic activity (acid or CO2, is in direct
proportion to the number of bacteria present).
10
Indirect method
The bacteria produce during metabolism end product like gas and these
products is compared to standard values (low number of
bacteria = 10 colonies or none at all)
The product of metabolism is direct proportion to number of bacteria
Ex: CO2 for a sample of bacteria is measured and compared to CO2
concentration of standard sample that contain low number
of bacteria or none
Dry weight
-For filamentous bacteria and molds, the fungus is
removed from the growth medium, filtered, and dried in a
desiccator. It is then weighed
Used usually for filamentous bacteria (bacteria between fungi and bacteria it
looks like fungi on plates but it is actually bacteria
The dry weight is indirect proportion with number of bacteria
Measuring Microbial Growth – Overview
Indirect Direct Method
Methods
Plate counts turbidity
MPN metabolic
activity
Direct microscopic count dry weight
Filtration
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Control of microbial growth
We talked previously about the chemical and physical factors to grow bacteria
here we will use these factors to kill, control and prevent
contamination
Alcohols and antibiotics are some of the chemical means to kill bacteria
Temperature is one of the physical factors, any increase in the temperature
might affect the bacteria to the point of killing it.(because
in our body we deal with mesophile bacteria)
Terminology
Sepsis: Characterized by the presence of pathogenic microbes in living
tissues or associated fluids. (Contaminated)
Asepsis: absence of significant contamination. (Not contaminated)
Aseptic surgery techniques prevent microbial contamination of wounds.
Antimicrobial: chemicals, expected to destroy pathogens but not to
achieve sterilization.
Disinfectant: used on objects (reduce the number of viable
microorganisms) kill viable cells only does not achieve sterilization. We use
alcohol as disinfectant and antiseptic as well.
Disinfectant تكون للطاولة مثال)تطهير( اماantiseptic فتكون مثال للجلد
Antiseptic: used on living tissue, destroys or inhibits the growth of
microorganisms does not achieve sterilization.
Nosocomial Infection (Hospital Acquired Infection) an infection that is
contracted from the environment or staff of a healthcare facility. (Mostly
acquired from the staff normal flora
12
Every person have normal flora and bacteria which he have biological
tolerance to so it does not cause any disease to him but it might cause
infection and be pathogenic to another person who does not have the same
normal flora and thus does not have biological tolerance to it
sepsisاخماج:
Anti: against
Sterilization: A defined process used to render a surface or product free
from viable organisms, including bacterial spores.
Sterilization: absence of any microorganisms (no viruses, no bacteria nothing
…)
Biocide: A chemical or physical agent, usually broad spectrum that
inactivates (kill) microorganisms.
include hydrogen peroxide, alcohols, bleach, Chemical biocides
cycloheximide, and phenols
include heat and radiation.biocides Physical
Fungicide, Virucide, Germicide, bactericide
-Cide = kill
- Broad spectrum = kills gram (-) and gram (+) and spore forming bacteria
-fungicide: kills fungi - virucide: kills viruses
- germicide: kills germs (general concept)
- Bactericide: kills bacteria
Sanitization: Lowering of microbial counts to prevent transmission in
public setting (e.g., restaurants & public rest rooms) lowering of microbial
counts to be accepted by public health regulation.
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Antibiotics: Naturally occurring and synthetically derived organic
compounds that inhibit or destroy selective bacteria, generally at low
concentrations
First antibiotics were natural but know mostly are synthetic.
Ex: penicillin.
We use antibiotic at low concentration because at high concentration it
becomes toxic.
There is two kind of antibiotics:
Bacteriostatic: Inhibits bacterial reproduction (affect one stage of bacterial
growth ex: nucleic acid and protein synthesis).
مثبطة وليست قاتلة
Bactericidal: Kills bacteria (ex: penicillin).
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Clostridium botulinum is kind of bacteria that cause severe food poisoning
(lethal)
Can lead to paralysis (disease called botulism).
In the production of canned food microbiologists take random samples
from canned food and put it in an incubator and check it after 2 weeks so
we decide if we put it in supermarkets or no.
Any bump in canned food would be caused by gases produced by bacteria.
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