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Streak Plate Method
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Go to simulatorObjective: 

 

  1. To obtain isolated microbial colonies from an inoculum by creating areas of increasing dilution on an agar petriplate.

 

Principle:

 

 

The streak plate method is a rapid qualitative isolation method. The techniques commonly used for isolation of discrete colonies initially require that the number of organisms in the inoculums be reduced. It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an agar plate. The resulting diminution of the population size ensures that, following inoculation, individual cells will be sufficiently far apart on the surface of the agar medium to effect a separation of the different species present. Although many type of procedures are performed, the four ways or quadrant streak is mostly done.

 

 

Importance of Streaking:

 

The human body has billions of bacteria which constitutes the normal flora fighting against the invading pathogens. It is tedious to isolate a particular type of bacteria from a clinical sample. Streak plate technique is used to grow bacteria on a growth media surface so that individual bacterial colonies are isolated and sampled. Isolated colonies indicate a clone of cells, being derived from a single precursor cell. When the selected culture media is inoculated using a single isolated colony, the resulting culture grows from that selected single clone. The modern streak plate method has evolved from the efforts by Robert Koch and other microbiologists to obtain pure culture of bacteria in order to study them. The dilution or isolation by streaking procedure was originally developed by Loeffler and Gaffky in Koch's laboratory, which  involves the dilution of bacteria by systematically streaking them over the surface of the agar in a petri dish to obtain isolated colonies which will subsequently grow into mass of cells, or isolated colonies. If the agar surface grows microorganisms which are all the genetically same, the culture is  then considered as a pure culture.

 

 The commonly used petri dishes are of hundred millimetre diameter. The agar surface of the plate should be dry without any moisture such as condensation drops. The source of inoculums can be clinical specimen, environmental swab, sedimented urine, broth or solid culture.

 

In the streaking procedure, a sterile loop or swab is used to obtain an uncontaminated microbial culture. The process is called "picking colonies" when it is done from an agar plate with isolated colonies and is transferred to a new agar or gelatin plate using a sterile loop or needle. The inoculating  loop or needle  is then streaked  over an agar surface. On the initial region of the streak, many microorganisms are deposited resulting in confluent growth or the growth of culture over the entire surface of the streaked area. The loop is sterilized by heating the loop in the blue flame of the Bunsen burner, between streaking different sections, or zones and thus lesser  microorganisms are deposited as the streaking progresses. The streaking process will dilutes out the sample that was placed in the initial region of the agar surface. There are two most commonly used streak patterns, a three sector "T streak " and a four quadrant streak methods.

 

Types of Media:

 

Growths of micro organisms require nutrients and environment conditions. Nutrient preparations made in laboratory, used for the growth of the organism are called media (singular: medium). Three physical forms are used: liquid or broth media; semisolid media; and solid media. Liquid media, such as nutrient broth, tryptic soy broth, or brain heart infusion broth, can be used to propagate large numbers of microorganisms in fermentation studies and for biochemical tests. Semisolid media can also be used in fermentation studies, in determining bacterial motility, and in promoting anaerobic growth. Solid media, such as nutrient agar or blood agar, are used for the surface growth of microorganisms in order to observe colony appearance, (2) for pure culture isolations, (3) lot storage of cultures, and (4) to observe specific biochemical reactions. While in the liquefied state, solid media can be poured into either a test tube or petri plate (dish) and if the agar is poured into a petri plate, the plate is designated an agar plate.

 

Media and Media Components:

 

Bacteria display a wide variety of nutritional and physical requirements for their growth. This includes water, a source of energy, sources of carbon, sulfur, nitrogen phosphorus, minerals such as Ca2+, Mg2+, Na+, and other vitamins and growth factors. Nutrient agar is a complex medium as it contains ingredients with unknown amounts or types of nutrients. Nutrient agar typically contains 0.5 % peptone, 0.3 % beef extract, 1.5 % agar in water (pH adjusted to neutral at 25 °C). Nutrient broth is made identically, omitting the agar.
 
Beef extract is the commercially prepared dehydrated form of autolysed beef and is provided in the form of a paste. Peptone source is casein (milk protein) that has been digested with the action of the enzyme pepsin. Peptone is dehydrated and supplied in the medium as powdered form. Peptone and beef Extract is a mixture of amino acids and peptides. Beef Extract also contains digest products which are water soluble, other macromolecules such as nucleic acids, fats, polysaccharides as well as vitamins and trace minerals (cannot be chemically defined). There are many ingredients in the media which are complex, which includes yeast extract, tryptone, and others. The importance of complex media is that they will support the growth of a wide range of micro organisms. Agar is obtained from red algae in which it is a supplement of polysaccharide (polygalacturonic acid) in their cell walls. Agar is not a  nutritional component, but  a better  solidification agent as  it dissolves at or near the  boiling temperature (100°C) but solidifies at 45°C. So molten (liquid) agar is prepared at 45°C, cells are mixed with it, and then allowed to solidify thereby trapping living cells.

 

 

Tryptic soy agar is the medium that supports the growth of most  of the fastidious organisms. Example: Streptococci, and some members of the genera Neisseria, Corynebacterium, Brucella, Listeria, Vibrio, Pasteurella, Erysipelothrix, etc. It is used to practice streaking technique and to observe differences in colony morphology;  for recovery of microorganisms from skin; to carry out the  catalase test and to test the requirements for X and/or V factors. E. coli is actually green on tryptic soy agar. The agar itself is a white, amber color.

 

Tryptic Soy Agar Formula Per Liter (Final pH 7.3 ± 0.2 at 25°C)

 

15 g of Tryptone(Pancreatic Digest of Casein ) , 5 g of Soytone (Papaic Digest of Soybean Meal ) , 5 g of Sodium Chloride, 15 g of Agar. Suspend 40 grams in 1 liter distilled or deionised water. Heat to boiling to dissolve completely. Autoclave at 121°C for 15 minutes. Cool to room temperature.

 

Media Sterilization:

 

Sterilizing media to above 1210C for 15 minutes in an autoclave destroys nearly all living cells and spores. If screw caped bottles are used, the cap must be loosened prior to sterilization process. Containers used for media preparation must possess vented tops and it must be adequate to hold at least 20% more than the assured volume of medium, to permit for the expansion during sterilization process. It is convenient to use 1L capped bottles for preparing large quantities. To promote cooling, handling, and pouring, we usually prepare them with 500 mL medium. Agar does not deliver uniformly when melted. To assure a uniform distribution for pouring into plates or tubes is to drop a magnetic stir bar in the flask or bottle, then stir the medium after sterilization gently, while the media cools. Stirring  spreads the agar evenly.

 

Pouring the Agar Plate:

 

The agar is immediately poured into a sterile, dry petri plate while holding the top carefully above the petri plate bottom in order to avoid contamination. Replace the top, allow the agar to cool and harden, and store the petri plates in an inverted position. When storing petri plates, do not stack them more than three high, or use a special petri plate storage holder. The agar is then poured into the plates using aseptic technique, preferably in a sterile cabinet (laminar air flow cabinet).

 

 

 

 

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