Microbiology sheet (6)

Made by marah marahleh

corrected by : abd. Salman

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

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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).

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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

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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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