CULTIVATION AND CULTIVATION AND IDENTIFICATION OFIDENTIFICATION OF

BACTERIABACTERIA

Doç.Dr.Hrisi BAHARDoç.Dr.Hrisi BAHARIstanbul UniversityIstanbul University

Cerrahpasa Medical Faculty Cerrahpasa Medical Faculty

WHAT IS CULTIVATION OF BACTERIAWHAT IS CULTIVATION OF BACTERIA

► The survival of microorganisms in the laboratory, as well as in nature, depends on their ability to grow under certain chemical and physical conditions

► Cultivation of bacteria is to obtain a bacterial growth under certain chemical and physical conditions .

BACTERIAL GROWTH

► An increase in population number of a bacteria by reproduction

► Not an increase in cell size

► Most bacteria reproduce by Binary Fission

◌ ◌ The cell doubles in size ◌ Replicate the chromosome ◌ Forms a septum in the center ◌ Synthesizes a cell wall at the septum ◌ Daughter cells separate

PURPOSE OF CULTURING BACTERIA

◌ ◌ Isolation of bacteriaIsolation of bacteria◌◌ Understand properties of bacteriaUnderstand properties of bacteria◌◌ To create antigen for laboratory useTo create antigen for laboratory use◌◌ To test for antibiotics sensitivityTo test for antibiotics sensitivity◌◌ Estimate viable countsEstimate viable counts◌◌ Maintain stock culturesMaintain stock cultures◌◌ Typing with bacteriophages and bacteriocins Typing with bacteriophages and bacteriocins

susceptibilitysusceptibility

A CULTURE MEDIAA CULTURE MEDIA

A culture culture mediamedia is a combination of nutrients used to growth organisms outside of their natural environment.

Culture media are employed in the

► Isolation and maintenance of pure cultures of bacteria

► Identification of bacteria according to their biochemical and physiological properties.

Major Contribution to Culture Media is provided by

Agar - Agar (Frau Hesse’s contribution)

Why we need culture media ?► Every organism must find in its environment

all of the substances required for energy generation and cellular biosynthesis.

► The chemicals and elements of this environment that are utilized for bacterial growth are referred to as nutrientsnutrients or nutritional requirementsnutritional requirements.

► In the laboratory, bacteria are grown in culture mediaculture media which are designed to provide all the essential nutrients in solution for bacterial growth.

Definitions Definitions

●● Pure CulturePure Culture : a single “strain” of microbe grown in media.●● StrainStrain: A microbial culture which is the

descendent of a single cell originally isolated from the environment.

●● Aseptic technique:Method of hendling material without contamination from the environment.

Classification of Culture Media According their productionAccording their production

► Synthetic mediaSynthetic media► Semi- Synthetic mediaSemi- Synthetic media► Non - Synthetic mediaNon - Synthetic media

According their usageAccording their usage

◌◌ MINIMAL MEDIUMMINIMAL MEDIUM

◌◌ ALL-PURPOSE MEDIUMALL-PURPOSE MEDIUM

◌◌ SELECTIVE MEDIUMSELECTIVE MEDIUM

◌◌ DIFFERENTIAL MEDIUMDIFFERENTIAL MEDIUM

A MINIMAL MEDIUMA MINIMAL MEDIUM

●● A minimal medium is one which supplies only the minimal nutritional requirements of a particular organism .

●● These media vary in composition according to the minimal nutritional requirements of the particular species under study

AN ALL- PURPOSE MEDIUM ●● This medium is rich in a wide variety of

nutrients (including many growth factors) and will, therefore, support the growth of a wide range of bacteria

◌◌ Chocolate AgarChocolate Agar

◌◌ Nutrient Agar Nutrient Agar

◌◌ Brain Heart Infusion AgarBrain Heart Infusion Agar

◌◌ Blood Agar Blood Agar

Blood culture – ‘Liquid Medium’

Chocolate agarChocolate agar

A SELECTIVE MEDIUM

●● This media supports the growth of desired organisms while inhibiting the growth of many or most of the unwanted ones

◌◌ MacConkey Agar.

◌◌ Salmonella,shigella Salmonella,shigella

mediummedium

Salmonella Shigella agarSalmonella Shigella agar

Lowenstein Jensen Medium - cultivation of Mycobacterium tuberculosis

A DIFFERENTIAL MEDIUM ●● This medium is one which allows two or more

different types of organisms to grow. It allows identification of bacteria based on

specific properties.

●● It contains dyes and/or other components upon which different organisms act in various ways to produce a variety of end products or effects, often detected by variations in color.

◌◌ Glucose Fermentation Broth ◌◌ Three sugar iron agarThree sugar iron agar

Three sugar iron Agar

Anaerobic Culture Methods Anaerobic jar

• Anaerobic jar

Figure 6.5

Basic requirements of culture mediaBasic requirements of culture media ► NutrientsNutrients

- Energy source - Carbon source - Nitrogen source

► Mineral saltsMineral salts – Sulphate, phosphates, chlorides & carbonates of K, Mg & Ca.

► A suitable pHA suitable pH – 7.2 – 7.4► Accessory growth factorsAccessory growth factors

- Tryptophan for Salmonella typhi - X & V factors for H. influenzae

Major elements, their sources and functions in bacterial cells.

According to physical properties, media can be classified as three types:

1. solid medium ( 1.5～ 2.5% of agar) agar plate

2. semi-solid medium (0.3～ 0.5% of agar)

3. liquid medium

1 2 31

Agar – AgarAgar – Agar

► Solid medium is made by adding aagargar

► Agar is obtained from Sea weeds of New Zealand

► Agar contain long chain of polysaccharides, Inorganic salts and protein like substances

► Melts at 980c and solidify at 420c

Mueller Hinton Agar for

Antibiotic Testing is a solid medium

Liquid Medium

► Difficult to identify all types of organisms

► Suitable for isolation of bacteria from Blood culturing or bacteria existing in small number.

IIDENTIFICATION OF BACTERIADENTIFICATION OF BACTERIA► The identification process started by isolating pure

colony by Quadrant streaking method on an appropriate culture media for the type of bacteria being targeted to be isolated.

This means:This means:

► To get suitable culture media that will allow the growth of certain species and inhibit others or

► To follow the standard and proper culturing method that enables us to isolate a pure colony.

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Quadrant streaking methode method

Figure 4.2

Identification of bacteriaIdentification of bacteria

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Identification of bacteriaIdentification of bacteria

●● Once we have got a pure colony the next step is to identify the bacterial species.

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Identification of bacteriaIdentification of bacteria

We start to accumulate information about the organism like:

► Morphological characteristics

► Culture characteristics and

► Physiological ( biochemical) characteristics

Bacterial Identification procedures

I- Microscopic proceduresII- Macroscopic procedures

• I- Microscopic proceduresI- Microscopic procedures ► Examination of unstained preparations (wet (wet mountmount examination examination )) for bacterial motility. ► Staining a preparation (Gram,E.ZN,GIEMSA)

Gram staining will determine:a- Bacterial Gram reactionb-Bacterial morphologyc- Bacterial strain arrangement

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Bacterial Identification procedures

• II- Macroscopic proceduresII- Macroscopic procedures

1- Gram reaction confirmation test1- Gram reaction confirmation test

• On a clean slide one or two colonies are mixed with two or three drops of 4% sodium hydroxide solution:

• Gram negative bacteria will produce a mucoid line when strengthen while gram positive will not produce it.

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Bacterial Identification procedures

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Identification of bacteriaIdentification of bacteria

2- Culture appearance2- Culture appearance : :

a- The appearance of bacterial colonies

b- The effect of colony on culture media are often characteristic of the bacterial species.

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Identification of bacteriaIdentification of bacteria

• a- The appearance of the colonies is described using conventional terms and may include:

– Shape of the colony

– Size of the colony

– Elevation of the colony

– Surface of the colony

– Color of the colony

– Opacity of the colony

– Consistency of the colony

Identification of bacteriaIdentification of bacteria

Identification need also to differentiate species following their aspects like:

1-Requirement of oxygen 2-The need of co2 3-Capacity to form pigments 4-Power of hemolysis

Identification of bacteriaIdentification of bacteria• b- The colony effects on culture media:

• 1- Pigment production :

• Pigment producing organisms will have colored colonies .The development of the color will depend on the type of the medium.

2-Beta hemolyses

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