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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.
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
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
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
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
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
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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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
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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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