Cultivation, Isolation, Purification And Characterization

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Amjad Khan Afridi
27/12/2018
General Methods Of Studying Microorganisms:
Cultivation, Isolation, Purification And Characterization
What are microbes?
A microorganism or Microbes is a microscopic living organism, which may be single-celled or
multicellular. The study of microorganisms is called Microbiology, subject that began with the
discovery of microorganisms in 1674 by Antonie Van Leeuwenhoek, using a microscope of his own
design.
Aseptic technique
• Aseptic technique refers to a procedure that is performed under sterile conditions.
• AIM: To prevent the access of micro-organisms during the preparation and testing.
In The Microbiology Lab We Use Aseptic Technique To:
• Prevent contamination of the specific microorganism we are working with.
• Prevent contamination of the room and personnel with the microorganism we are working with.
Sources OfContamination
1. The Atmosphere
2. The Breath
3. The Hands
4. Clothing
5. The Hair
6. The Working Surface
7. Equipment
Sample Collection for the isolation ofMicrobes
Different samples can be collected by using different techniques .
Different types of samples are using for the isolation of microbes.
Examples
1. Clinical Samples
2. Soil Samples
3. Water Samples
a. Fresh Water Samples
b. Waste Water Samples

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Amjad Khan Afridi
27/12/2018
4. Food Samples
5. Milk Samples
a. pasteurized Milk
b. Unpasteurized Milk
6. Air etc…
1. Clinical Samples
Clinical sample should be collected from the active site of infection and precautions should be taken to
exclude surface contamination of the sample.
We can use different body fluids or tissue of the body for the isolation of the microbes.
Body fluids , i.e :
Blood, Urine, Saliva, Pus, Sputum, CSF (Cerebrospinal fluid), Stool, Semen, Synovial fluid, Pericardial
fluid, Peritoneal fluid, Pleural fluid, Vaginal discharge and body Tissue etc.
Culture
What is a culture?
Population of microorganisms grown under well defined conditions. i.e
 Nutrients
 Temperature
 pH
 Humidity
 Time ( Incubation period )
The specimens received in the laboratory and Streaking on the plated culture media. The appropriate
culture media is selected depending upon the bacteria suspected. The following precautions need to be
taken into consideration when the culture methods are processed.
 Optimal atmospheric conditions ( To provide specific Incubation period )
 Optimal temperature (Most of the bacteria requires a temperature of 37°C for optimal growth)
 Growth requirement of the bacteria (Different bacteria have different growth requirements)
What is mixed culture?
When a particular species of microbes is present in a very small number in comparison to the total
number of microorganisms, such culture is called as mixed culture.

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Amjad Khan Afridi
27/12/2018
What is pure culture?
A culture containing only one species of microbe is called pure culture.
Species: A collection of bacterial cells which share an overall similar pattern of traits in contrast to other
bacteria whose pattern differs significantly.
Strain: A strain is a subset of a bacterial species differing from other bacteria of the same species by
some minor but identifiable difference.
Medium
Medium is the plural of media.
Different types of medium are use for the bacterial culturing .
Medium forms
Have two forms
1. Liquid ( broth ) media . (Liquid media are used for growth of pure batch cultures)
2. Solid media ( semi-solid). (solidified media are used widely for the isolation of pure cultures, for
estimating viable bacterial populations, and a variety of other purposes.
Have two types
1. Selective media ( support / growth specific microbe)
2. Differential media ( Can support & growth both gram positive & negative bacteria)
Names of some medium used for bacterial and fungus culturing.
 Nutrient Agar
 EMB ( Eosin Methylene Blue)
 MecConkey Agar
 Blood Agar
 Chocolate Agar
 Mannitol Salt Agar
 Pepton Water
 PDA ( Sabouraud Detose Agar)
 Dermatophyte Agar
 Sabouraud Liquid Medium etc.
Streaking
Streaking is a technique used to isolate a pure strain from a single species of microorganism, often
bacteria & fungi. Sample can be taken from the resulting colonies and a microbiological culture can be
grown on a new plate, so that the organism can be identified, studied, or tested.

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Amjad Khan Afridi
27/12/2018
This method is used most commonly to isolate pure cultures of bacteria. In This method the tip of a fine
structure wire loop called Inoculation needle consist of a wooden or glass handle with a nichrome wire
the end of which is bend to form a loop is used to transfer microbes from culture. The straight wires are
similar to wire loop except they do not have loop. These are used to transfer culture in colony formed on
solid culture medium. In such cases, the colony from solid medium is streaked on the surface of nutrient
agar medium in a sterile petri-dish.
Streak- Plate Method
 This technique consists of the following steps:
 Hold the broth culture containing tube in left hand and shake it.
 Sterilize the wire loop of the inoculation needle on burner flame .
 Remove the cotton plug of the broth culture tube by little finger of right hand.
 Flame the mouth of the test tube immediately.
 Insert the wire loop to form a thin film and replace the cotton plug.
 The thin film in the loop is streaked in either a zig-zag manner by removing the loop backwards
and forwards firmly. Care should be taken that loop should not be firmly pressed against the agar
surface.
 Incubate the petri dish in incubator at a required temperature.
 Growth of the bacteria will be visible (after an overnight incubation)on the streaked marks.
Incubation
Allow the cultured plate in the incubator at 37°C for 24 hrs for growth.
After 24 hrs observe the growth of bacteria / fungi on cultured plate.
For fungi apply 28°C for 24 hrs to 1 week or more then this, depending on the natural growth period of
fungi.
Isolation
The process of screening a pure culture by separating one type of microbes from a mixture is called
Isolation.

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Amjad Khan Afridi
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Microorganisms are generally found in nature (air, soil and water) as mixed populations. Even the
diseased parts of plants and animals contain a great number of microorganisms, which differ markedly
from the microorganisms of other environments. To study the specific role played by a specific
microorganism in its environment, one must isolate the same in pure culture..
Purification
What is pure culture?
A culture containing only one species of microbe is called pure culture.
Importance
 To isolated a single colony.
 To isolated desire colony.
 To get a single colony/ species from a mixed growth culture.
In this process a growth culture medium is prepared, a single bacterial colony is picked from growth
plate by using sterile wire loop and streak on the culture plates.
These culture plates will keep in the incubator at 37°C for 24 hrs, for growth.
After 24 hrs, billions colonies of a single bacterial strain will appear on the culture plates.
A pure culture theoretically contains a single bacterial species. There are a number of
procedures available for the isolation of pure cultures from mixed populations. A pure culture
may be isolated by the use of special media with specific chemical or physical agents that allow
the enrichment or selection of one organism over another. The differential and selective
procedures will be utilized later in this course. Simpler methods for isolation of a pure culture
include: (i) spread plating on solid agar medium with a glass spreader and (ii) streak plating with
a loop. The purpose of spread plating and streak plating is to isolate individual bacterial cells
(colony-forming units) on a nutrient medium.
Both procedures (spread plating and streak plating) require understanding of the aseptic
technique. Asepsis can be defined as the absence of infectious microorganisms. However, the
term is usually applied to any technique designed to keep unwanted microorganisms from
contaminating sterile materials.
Characterization & Identification
1. Morphological Identification
Bacteria grow tremendously fast when supplied with an abundance of nutrients. Different types of
bacteria will produce different-looking colonies, some colonies may be colored, some colonies are
circular in shape, and others are irregular. The characteristics of a colony (shape, size, pigmentation, etc.)
are termed the colony morphology.

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Amjad Khan Afridi
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Many fungi produce colonies with a floppy appearance similar to cotton wool. The molds produce
colonies which on aging develop a dry chalky appearance.
Microscopy
A Gram stain helps with the visualization of bacteria, and gives an indication of the type of bacteria
present, based on the shape of the bacteria and the staining properties (Gram positive: purple; Gram
negative: pink/red). A Gram stain also helps to identify mixtures of bacteria, helps to determine the
appropriate range of agar plates to be used for subsequent culture, and helps with the interpretation of
culture results.
2. Biochemical Identification
Different types of biochemical test are perform for the identification of gram (+) and gram (-) bacteria .
i.e. Catalase,Coagulase, Urease,Oxidase Activities, TSI, carbohydrate fermentation and Indole
production tests.