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Microbiology sheet (5)

Lecture:

Created by: marah hiary & tamara shwabka

Corrected by: abd. salman & nadeen hadidi

Date: 4/10/2016

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: Some notes about the previous lecture

*One different between eukaryote and prokaryote cell that the prokaryote has one

single circular chromosome while eukaryote has more than one chromosome.

* Most of the bacteria have one circular chromosome.

*few bacteria has more than one linear chromosome.

*number of chromosome or nucleoid depend on growth condition or environmental

conditions.

*sometimes according to environmental and growth conditions the bacteria can't

divide (by binary fission) this cause to have more than one nucleoid and this is not

normal event.

*there is no 100% percent in microbiology.

Microbial growth

*70% of infectious bacteria has biofilm.

*it resists chemical and physical factor and antibiotic 100x than other bacteria. And it

gets nutrient, remove waste.

* Quorum sensing: when bacteria reach threshold value (maximum number) it will

produce auto inducer molecule this will bind to receptor this binding will switch on

virulence gene (toxin gene).

*نظام استشعار يعتمد على الكثافة العددية للبكتيريا.

لها القصى قيمة وبالتالي لن ينتج وصول الكثافة العددية نمنع و*اذا اوقفنا هذه العملية سوف نوقف نمو البكتيريا

auto inducer molecule هذا ما تستخدمه بعض التكنولوجيا الحديثة.و ولن يحدث تشغيل للجينات الممرضة

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Biofilm:

Microbial communities used slime for attachment of bacterium to surface

structures or to each others. Bacteria communicate by chemicals via quorum

sensing which is a system of stimuli and response correlated to population

density

Facilitating the transfer of genetic information's (conjugation.)

Slime Sheltered the bacteria from harmful factors (such as desiccation,

antibiotics, and the body's immune system.)

Slime producing bacteria is the cause of most nosocomial infections

Nosocomial infections=hospital acquired infections.*

Example: pseudomonas (normal flora: exist in our body naturally so it doesn’t cause

diseases) exist in skin, GIT, eye drops, ear drops, washing solutions

Resist 40 type of antibiotics.

Culture media

*The goal of the development of bacteria (doing cultivate) is to become in the form

of colonies and thus facilitate the identification of its kind.

*Culture media has different color and there is some media for cultivate gram + and

other for gram – and other selective for fastidious bacteria

s.has a complex nutritional requirement*fastidious bacteria:

))تحتاج متطلبات نمو عالية

*we use wire loop for cultivating.

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*bacteria need 18-24 hours to form colonies.

*2 types of media:

1) Chemically defined media: this mean that the concentrations of all component of

the media are well-defined.

2) Complex media (used in the lab): made of nutrients (concentrations are not

defined)

*the different between Nutrient broth Nutrient agar

Solid, agar , no agar liquid (suspension)

*agar is polysaccharide used for solidification.

*Why don’t we use starch instead of agar??

Answer: we don’t use starch for cultivation because bacteria has amylase enzyme

that dissociate starch so the media will be liquid while it hasn't enzyme for agar.

* Temperature suitable for housing in the dishes 50-60

) we obtain agar from algae*الطحالب البحرية (

•Culture medium: A nutrient material prepared for the growth of microorganisms

in a laboratory. Should be sterile (not contain living microbes)

•Inoculum: Microbes that are introduced into a culture medium to initiate growth

•Culture: Microbes growing and multiplying in/on culture medium

•Chemically defined media: is one whose exact chemical composition is known. (For

research purposes only)

•Complex media: made up of nutrients including extracts from

•yeasts, meat, or plants, or digests of proteins from these and

•other sources. The exact chemical composition varies slightly

•from batch to batch. E.g. Nutrient broth, Nutrient agar, Blood agar

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*we can find E.coli in intestine

Agar :

•Complex polysaccharide

•Used as solidifying agent for culture

media in Petri plates, and slants

•Generally not metabolized

by microbes

•Liquefies at 100°C

•Solidifies ~40°C

*types of culture Media according to composition and uses:

1) Selective medium: certain substances (it enhances some microorganisms and

inhabits other microorganisms)

Ex: MacConkey selective for gram –

- gramالن المسبب لها في حاالت التهاب المسالك البولية نستخدمها

mannitol salt agar selective for gram +

2) Differential medium: differentiate between two types of bacteria.

The same media can be selective and differential at the same time. *

*hemolysis: dissociation of red blood cells.

*beta hemolysis: completely dissociation of red blood cell

(most pathogenic) حول البكتيريا و تظهر المنطقة شفافة

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*Alfa hemolysis: partially dissociation of red blood cell and its greenish to black color

because of release of hemoglobin from RBC

* Gama hemolysis: means no hemolytic activity.

Enrichment medium

•Designed to increase numbers of desired microbes to detectable levels

•e.g. Blood agar

•Used for fastidious bacteria

Simple medium

•Provide the minimal requirements for bacterial growth

•e.g. Nutrient agar

*simple medium: contains all requirements for growth of all types of bacteria.

الن جميع انواع البكتيريا تظهر متشابهة بالشكل و بالتالي ال نستطيع تميزها في تفريق البكتيريا*ال نستفيد منه

*The process of identification of the bacteria takes from 48-72 hours.

*after the identification we have to make the sensitivity test (sensitivity to

antibiotics) and as we said we can’t identify the bacteria by simple medium so we

have to use selective medium.

Pure culture: Contain only one species or strain.

Most patient specimens and environmental samples contain several different kinds

of bacteria (mixed culture)

•Streak-plate method: Most bacteriological work requires pure cultures, or clones,

of bacteria. Streaking is the isolation method most commonly used to get pure

cultures

•Colony formation: A population of cells originated from a single cell or spore or

from a group of attached cells (also referred to as CFU= colony forming unit.)

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•Aseptic technique: is a method designed to prevent contamination from

microorganisms

Culture need 2 day

Sensitive need 1 day

*pure culture: one type of bacteria can found it has the same color, size, smell, edge

…….. (Same appearance)

*mix culture: more than one type of bacteria and it has different color, size, edge ……

When we take a sample of the urine of a patient and cultivate it, this sample is mix

culture because in addition to the bacteria that causes the disease we have normal

flora.

*ال نستطيع استخدامها لفحص الحساسية النها قد تكون واحدة حساسة و واحدة مقاومة و اليمكن تحديدها

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*we have to dilute the sample.

*we make a zigzag to increase number of bacteria

*95% of UTI caused by e-coli.

:UTIالتهاب المسالك البولية

اال ان وجودها في المسالك البولية large intestine في ال normal floraعبارة عن e-coli بالرغم ان ال

يسبب في حدوث التهاب.

والمسالك البولية حيث المسافة بينهما قليلة جدا. anal canalان تنتقل بين ال e-coli*يمكن لل

بيئة مناسبة لنمو البكتيريا. urine *ال

Bacterial reproduction • Binary fission: used for bacterial reproduction

• Budding: A few bacterial species reproduce by budding; a small initial outgrowth (a bud) that enlarges until its size approaches that of the parent cell, and then it separates.

•A few filamentous species simply fragment, and the fragments initiate the growth of new cells. •Generation time – time required for cell to divide (doubling time). Ranges from 20 min (E. coli) to > 24h (M. tuberculosis)

*pathogenic bacteria has a short generation time.

*one million cells of e-coli need 20 minutes to become 2 million (generation time=20

minutes)

*number of e-coli cells that cause UTI must be more than 100000.

*vibrio cholera can’t be normal flora.

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Number of bacteria = 2^n

:Nاالنقساماتعدد

Q. If a single E. coli cell reproduced every 20 minutes, how many Would there be in 6 hours?

Answer: 2^18 cells

n= (60 minutes*6 /20 minutes (generation time))

(Slide 22) *Bacteria have their own stages of growth .

*All kinds of bacteria pass in 4 stages :

1)the lag phase ( الطور التمهيدي) It’s the first stage ,when bacteria divide they should duplicate their

ribosomes and nuclear material so everything will be doubled .

* in this phase the cell is not dormant : its metabolismy active so

it can reaches to the peak .

*A straight line is the way of the division process .

The lag phase - This period of little or no cell division. During this time, however, the cells are not dormant. The microbial population is undergoing a period of intense metabolic activity involving, synthesis of enzymes and various molecules

The log phase (exponential phase) - The cells begin to divide and enter a period of growth, or logarithmic increase. Cellular reproduction is most active during this period.

2)exponential or logarithmic stage (log phase ) (الطور التصاعدي) :

-Begin the process of division .

-period of division is the most important period in bacterial life .

3) the stationary stage :

appears as a straight line . ( طور الثبات و السكون)

the number of dividing cells = the number of dyeing cells.

No increase or decrease in total cells number and thus be a

straight line.

(Slide 23)

The stationary phase - The growth rate slows, the number of microbial deaths balances the number of new cells, and the population stabilizes. (period of equilibrium)

What causes exponential growth to stop is not always clear. 1.The exhaustion of nutrients 2.The accumulation of waste products, and harmful changes in ph may all play a role.

3)the death phase :

increase the population of dead cells to become the top of the

growing cells.

note that the bacteria die gradually.

(slide 21 )

Bacterial Growth Curve Phases of growth

–Lag phase

The death phase - The number of deaths eventually exceeds the number of new cells formed, and the population enters the death phase, or logarithmic decline phase.

–Exponential or logarithmic (log) phase (growth phase )

–Stationary phase

–Death phase (decline phase)

ànote : there is deference in period of time in each phase .

**Note : ي المختير لمدة اسبوع او اقل

ىض البقاء ف يا فمن المفير اذا أردنا رسم المنحنى للبكتير

يا وتسجيل اعددادها كل ق حيث تقاس عن دقائ 5حنر نستطيع مراقبة نمو البكتيرطريق جهاز يسىم

) Spectrophotometer (

ة الزمنية لكل مرحلة يتم التسجيل اىل ان تثبت القراءة ومن ثم يتم حساب الفير

àIn industries it does not useful use bacteria from the first phase ,

why ?

it is not useful to use bacteria from ( lag) phase because it needs a

period of time to divide , as we know all of the metabolism

process occur in the (log ) phase so we use Bactria from this

phase and ferment them like putting them in a special container

which can enter the nutrient from one side and exit the west

product from the other side .....which known as "continuous

culture" .

(Slide 25)

-How to measure the microbial growth ?

there are direct and indirect ways .

-direct ways plate counts and it have two methods (the

pour plate method , the spread plate method) And In these two

methods we must make dilution .

for example if we have milk or yoghurt sample and it contains

a contamination, how do we calculate the number of bacteria ?

A- dilution :

Measurements of Microbial Growth Direct Methods Viable cell counts: Plate counts: Serial dilutions put on

1)Bring 6 test tubes at least and put 9 mil of normal saline in each

.

2)add 1 ml from sample.

Why do we use normal saline ?

Because normal saline (Isotonic) so it do not affect the bacteria

-after adding :

Total Volume = 9ml+1ml =10 ml

Dilution =

X = المادة المراد تخفيفها قبل التخفيف . D= المادة المخفف بها

-dilution at (test tube 1)=

=

=

= 10^-1

Then we will take 1ml from the first test tube and dilute it in the

second one so the dilution at the second one is =10^-2

And then from second one to third one so dilution at third = 10^-

3 … we will repeat it until we finish the sixth one = 10^-6 .

Note that every turn we take 1ml from previous test tube and the

total volume for each test tube is 9ml except the sixth is 10ml

because every time we going to take 1ml from the previous one

so ( 10-1=9 ml) but the sixth is the last so it will be 10ml .

Ex: In the second test tube =1/10 X1/10 =1/100 = 10^2 and

ext.….

B-NOW WE CAN USE ANY OF DIRECT METHODS : 1)The pour plate method : A-inoculate an empty plate (in this test we will take 6 because we

use 6 test tubes)

B-add melted ager

(add 1ml from each dilution and mix it with 13 ml of melted ager

)

Increase in dilution means decrease of concentration of bacteria.

-agar at 55c is liquid not solid

the principle of calculating the number of bacteria depends on

dilution .

we take a plate that contains the bacteria in between (30 -300)

to use it in measurement .

bacteria with number less than 30 will give an error .

Bacteria with number more than 300 will be difficult to

calculate .

àColonies in this way appear :

A) at surface

b) on subsurface

CFU/ML =NO. x DF If we took the fifth one for example and it had 50 colonies so the

total colonies number per ml will be :

Cfu= 50 X 10^5 = 5,000,000

We take DF in its positive form .

2)the spread plate method : Inoculate plate containing solid medium (containing agar )

Here instead 1ml we will take 0.1ml from each test tube and

mix it with agar . So

CFU /ML= NO X DF X10

We multiply it by 10 because its per 1ml not 0.1ml.

and Here the colonies are at the surface only because we use

the spread sheet.

Note :some details I wrote it like how I did in the lecture because

numbers and calculations in the record aren't clear enough to

understand so it can't be 100% like what you goanna to listen . Nadeen hadidi