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As stated in Lab 2, microorganisms exist in nature as mixed populations. Two major steps are involved in obtaining pure cultures from a mixed population:
- First, the mixture must be diluted until the various individual microorganisms become separated far enough apart on an agar surface that after incubation they form visible colonies isolated from the colonies of other microorganisms. This plate is called an isolation plate.
- Then, an isolated colony can be aseptically "picked off" the isolation plate (Figure 1) and transferred to new sterile medium (see Fig. 3). After incubation, all organisms in the new culture will be descendants of the same organism, that is, a pure culture.
Figure 1: Picking a Single Colony 0ff of a Petri Plate in order to Obtain a Pure Culture Before removing bacteria from the petri plate, first cool the loop by sticking it into the agar away from any growth.
A. STREAK PLATE METHOD OF ISOLATION
The most common way of separating bacterial cells on the agar surface to obtain isolated colonies is the streak plate method we used in Lab 2 to inoculate a petri plate. It provides a simple and rapid method of diluting the sample by mechanical means. As the loop is streaked across the agar surface, more and more bacteria are rubbed off until individual separated organisms are deposited on the agar. After incubation, the area at the beginning of the streak pattern will show confluent growth while the area near the end of the pattern should show discrete colonies (see Fig. 2A and Fig. 2B).
B. THE POUR PLATE AND SPIN PLATE METHODS OF ISOLATION
Another method of separating bacteria is the pour plate method. With the pour plate method, the bacteria are mixed with melted agar until evenly distributed and separated throughout the liquid. The melted agar is then poured into an empty plate and allowed to solidify. After incubation, discrete bacterial colonies can then be found growing both on the agar and in the agar.
The spin plate method involves diluting the bacterial sample in tubes of sterile water, saline, or broth. Small samples of the diluted bacteria are then pipetted onto the surface of agar plates. A sterile, bent-glass rod is then used to spread the bacteria evenly over the entire agar surface (see Fig. 4) in order to see isolated colonies (see Fig. 5). In Lab 4 we will use this technique as part of the plate count method of enumerating bacteria.
C. USE OF SPECIALIZED MEDIA
To supplement mechanical techniques of isolation such as the streak plate method, many special-purpose media are available to the microbiologist to aid in the isolation and identification of specific microorganisms. These special purpose media fall into four groups: selective media, differential media, enrichment media, and combination selective and differential media.
1. Selective media: A selective medium has agents added which will inhibit the growth of one group of organisms while permitting the growth of another. For example, Columbia CNA agar has the antibiotics colistin and nalidixic acid added which inhibit the growth of Gram-negative bacteria but not the growth of Gram-positives. It is, therefore, said to be selective for Gram-positive organisms, and would be useful in separating a mixture of Gram-positive and Gram-negative bacteria.
2. Differential media: A differential medium contains additives that cause an observable color change in the medium when a particular chemical reaction occurs. They are useful in differentiating bacteria according to some biochemical characteristic. In other words, they indicate whether or not a certain organism can carry out a specific biochemical reaction during its normal metabolism. Many such media will be used in future labs to aid in the identification of microorganisms.
3. Enrichment media: An enrichment medium contains additives that enhance the growth of certain organisms. This is useful when the organism you wish to culture is present in relatively small numbers compared to the other organisms growing in the mixture.
4. Combination selective and differential media: A combination selective and differential medium permits the growth of one group of organisms while inhibiting the growth of another. In addition, it differentiates those organisms that grow based on whether they can carry out particular chemical reactions.
For example, MacConkey agar (see Fig. 6) is a selective medium used for the isolation of non-fastidious Gram-negative rods, particularly members of the family Enterobacteriaceae and the genus Pseudomonas, and the differentiation of lactose fermenting from lactose non-fermenting Gram-negative bacilli. MacConkey agar contains the dye crystal violet well as bile salts that inhibit the growth of most Gram-positive bacteria but do not affect the growth of most Gram-negatives. If the Gram-negative bacterium ferments the sugar lactose in the medium, the acid end products lower the pH of the medium. The neutral red in the agar turns red in color once the pH drops below 6.8. As the pH drops, the neutral red is absorbed by the bacteria, causing the colonies to appear bright pink to red.
Results are interpreted as follows:
- Strong fementation of lactose with high levels of acid production by the bacteria causes the colonies and confluent growth to appear bright pink to red. The resulting acid, at high enough concentrations, can also causes the bile salts in the medium to precipitate out of solution causing a pink precipitate (cloudiness) to appear in the agar surrounding the growth (see Fig. 7).
- Weak fermentation of lactose by the bacteria causes the colonies and confluent growth to appear pink to red, but without the precipitation of bile salts there is no pink precipitate (cloudiness) in the agar surrounding the growth (see Fig. 8).
- If the bacteria do not ferment lactose, the colonies and confluent growth appear colorless and the agar surrounding the bacteria remains relatively transparent (see Fig. 9).
Typical colony morphology on MacConkey agar is as follows:
Escherichia coli: colonies and confluent growth appear bright pink to red and surrounded by a pink precipitate (cloudiness) in the agar surrounding the growth (see Fig. 7).
Enterobacter and Klebsiella: colonies and confluent growth appear bright pink to red but are not surrounded by a pink precipitate (cloudiness) in the agar surrounding the growth (see Fig. 8).
Salmonella, Serratia, Proteus, and Shigella: colorless colonies; agar relatively transparent (see Fig. 9).
There are literally hundreds of special-purpose media available to the microbiologist. Today we will combine both a mechanical isolation technique (the streak plate) with selective and selective-differential media to obtain pure cultures from a mixture of bacteria. In future labs, such as 12 - 16, which deal with the isolation and identification of pathogenic bacteria, we will use many additional special-purpose media.
One plate of each of the following media: Trypticase Soy agar, Columbia CNA agar, and MacConkey agar.
A broth culture containing a mixture of one of the following Gram-positive bacteria and one of the following Gram-negative bacteria:
- Possible Gram-positive bacteria:
- Micrococcus luteus. A Gram-positive coccus with a tetrad or a sarcina arrangement; produces circular, convex colonies with a yellow, water-insoluble pigment on Trypticase Soy agar.
- Micrococcus luteus growing on TSA
- Close up of Micrococcus luteus growing on TSA
- Staphylococcus epidermidis. A Gram-positive coccus with a staphylococcus arrangement; produces circular, convex, non-pigmented colonies on Trypticase Soy agar.
- Staphylococcus epidermidis growing on TSA
- Close up of Staphylococcus epidermidis growing on TSA
- Micrococcus luteus. A Gram-positive coccus with a tetrad or a sarcina arrangement; produces circular, convex colonies with a yellow, water-insoluble pigment on Trypticase Soy agar.
- Possible Gram-negative bacteria:
- Escherichia coli. A Gram-negative bacillus; produces irregular, raised, non-pigmented colonies on Trypticase Soy agar.
- Escherichia coli growing on TSA
- Enterobacter aerogenes. A Gram-negative bacillus; produces irregular raised, non-pigmented, possibly mucoid colonies on Trypticase Soy agar.
- Enterobacter aerogenes growing on TSA
- Escherichia coli. A Gram-negative bacillus; produces irregular, raised, non-pigmented colonies on Trypticase Soy agar.
During the next three labs you will attempt to obtain pure cultures of each organism in your mixture and determine which two bacteria you have. Today you will try to separate the bacteria in the mixture in order to obtain isolated colonies; next lab you will identify the two bacteria in your mixture and pick off single isolated colonies of each of the two bacteria in order to get a pure culture of each. The following lab you will prepare microscopy slides of each of the two pure cultures to determine if they are indeed pure.
PROCEDURE (to be done in pairs)
1. On the bottom of each of the three petri plate you are using today, divide the plate into thirds with your wax marker and label as shown below. This will guide your streaking.
2. Although Trypticase Soy agar (TSA), which grows both Gram-positive and Gram-negative bacteria, is not normally used as an isolation medium, we will attempt to obtain isolated colonies of the two organisms in your mixture by using strictly mechanical methods. Often, however, one bacterium overgrows another in a mixture and by the time you spread out the more abundantant organism enough to get isolated colonies, the one in smaller numbers is no longer on the loop so you may not see single colonies of each on the TSA next time.
Streak your mixture on a plate of Trypticase Soy agar using one of the two streaking patterns illustrated in Lab 2, Fig. 4 and Fig. 5. agar
3. Streak the same mixture for isolation (see Fig. 5) on a plate of Columbia CNA agar (selective for Gram-positive bacteria).
- Micrococcus luteus growing on Columbia CNA agar.
- Staphylococcus epidermidis growing on Columbia CNA agar.
4. 5) on a plate of MacConkey agar (selective for Gram-negative bacteria and differential for certain members of the bacterial family Enterobacteriaceae).
- Escherichia coli growing on MacConkey agar.
- Enterobacter aerogenes growing on MacConkey agar.
5. Incubate the three plates upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period.
1. Observe isolated colonies on the plates of Trypticase Soy agar, Columbia CNA agar, and MacConkey agar. Record your observations and conclusions.
Trypticase Soy agar
Columbia CNA agar
2. Using any of the three plates on which they are growing:
a. Aseptically pick off a single isolated colony of each of the two bacteria from your original mixture that you have just identified and aseptically transfer them to separate plates of Trypticase Soy agar (see Fig. Remember to streak the plate for isolation as you learned in labs 2 and 3.
b. When picking off single colonies, remove the top portion of the colony without touching the agar surface itself to avoid picking up any inhibited bacteria from the surface of the agar. Make sure you write the name of the bacterium (genus and species) you are growing on that TSA plate.
c. Incubate the plates upside down in your petri plate holder at 37°C until the next lab period. These will be your pure cultures for Lab 5 (Direct and Indirect stains).
PERFORMANCE OBJECTIVES FOR LAB 3
After completing this lab, the student will be able to complete the following objectives:
1. Given a mixture of a Gram-positive and a Gram-negative bacterium and plates of Columbia CNA, MacConkey, and Trypticase Soy agar, describe the steps you would take to eventually obtain pure cultures of each organism.
2. Define: selective medium, differential medium, enrichment medium, and combination selective-differential medium.
3. State the usefulness of Columbia CNA agar and MacConkey agar.
4. Describe how each of the following would appear when grown on MacConkey agar:
a. Escherichia coli
b. Enterobacter aerogenes
1. Using the streak plate method of isolation, obtain isolated colonies from a mixture of microorganisms.
2. Pick off isolated colonies of microorganisms growing on a streak plate and aseptically transfer them to sterile media to obtain pure cultures.
1. When given a plate of Columbia CNA agar or MacConkey agar showing discrete colonies, correctly interpret the results.
Lab 3: Obtaining Pure Cultures from a Mixed Population - Biology
Isolation of Pure Culture
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. Pure culture involves not only isolation of individual microorganisms from a mixed population, but also the maintenance of such individuals and their progenies in artificial media, where no other microorganisms find way to grow.
However, it is not easy to isolate the individual microorganisms from natural habitats and grow them under imposed laboratory conditions. For this, great deal of laboratory manipulation is required. If inoculums from any natural habitat is taken and allowed to grow in a culture medium, a large number of diverse colonies may develop that, due to crowdedness, may run together and, thereby, may lose individuality. Therefore, it is necessary to make the colonies well-isolated from each other so that each appears distinct, large and shows characteristic growth forms. Such colonies may be picked up easily and grown separately for detailed study. Several methods for obtaining pure cultures are in use. Some common methods are in everyday-use by a majority of microbiologists, while the others are methods used for special purposes.
Common Methods of isolation of pure culture
Pure culture of microorganisms that form discrete colonies on solid media, e.g., yeasts, most bacteria, many other microfungi, and unicellular microalgae, may be most commonly obtained by plating methods such as streak plate method, pour plate method and spread plate method.
But, the microbes that have not yet been successfully cultivated on solid media and are cultivable only in liquid media are generally isolated by serial dilution method.
Streak Plate Method
This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the surface of the agar medium. The successive streaks "thin out" the inoculums sufficiently and the microorganisms are separated from each other. It is usually advisable to streak out a second plate by the same loop/needle without reinoculation. These plates are incubated to allow the growth of colonies. The key principle of this method is that, by streaking, a dilution gradient is established across the face of the Petri plate as bacterial cells are deposited on the agar surface. Because of this dilution gradient, confluent growth does not take place on that part of the medium where few bacterial cells are deposited
Various methods of streaking
Presumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture. Such isolated colonies are picked up separately using sterile inoculating loop/ needle and restreaked onto fresh media to ensure purity.
Pour Plate Method
This method involves plating of diluted samples mixed with melted agar medium. The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium. Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state at a temperature of 42-45°C (agar solidifies below 42°C).
The bacteria and the melted medium are mixed well. The contents of each tube are poured into separate Petri plates, allowed to solidify, and then incubated. When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies). These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure purity.
Pour plate method has certain disadvantages as follows: (i) the picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, and (ii the microbes being isolated must be able to withstand temporary exposure to the 42-45° temperature of the liquid agar medium therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms.
However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture.
B. Pouring of the plate and
C. Colony development after incubation. Control consists of the sterilized plating medium alone
The isolated colonies are picked up and transferred onto fresh medium to ensure purity. In contrast to pour plate method, only surface colonies develop in this method and the microorganisms are not required to withstand the temperature of the melted agar medium.
Spread Plate Method
In this method the mixed culture of microorganisms is not diluted in the melted agar medium (unlike the pour plate method) it is rather diluted in a series of tubes containing sterile liquid, usually, water or physiological saline. A drop of so diluted liquid from each tube is placed on the centre of an agar plate and spread evenly over the surface by means of a sterilized bent-glass-­rod.
The medium is now incubated. When the colonies develop on the agar medium plates, it is found that there are some plates in which well-isolated colonies grow. This happens as a result of separation of individual microorganisms by spreading over the drop of diluted liquid on the medium of the plate.
Serial Dilution Method
As stated earlier, this method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media. A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions.
The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution. The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that there are some tubes showing growth of only one individual microbe. For convenience, suppose we have a culture containing 10 ml of liquid medium, containing 1,000 microorganisms i.e., 100 microorganisms/ml of the liquid medium.
Serial dilution method
If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would then have 100 microorganisms in 10 ml or 10 microorganisms/ ml. If we add 1 ml of this suspension to another 9 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism. If this tube shows any microbial growth, there is a very high probability that this growth has resulted from the introduction of a single microorganism in the medium and represents the pure culture of that microorganism.
Special Methods of Isolation on of Pure Culture
1. Single Cell Isolation methods
An individual cell of the required kind is picked out by this method from the mixed culture
and is permitted to grow. The following two methods are in use.
(i) Capillary pipette method
Several small drops of a suitably diluted culture medium are put on a sterile glass-coverslip by a sterile pipette drawn to a capillary. One then examines each drop under the microscope until one finds such a drop, which contains only one microorganism. This drop is removed with a sterile capillary pipette to fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure culture.
(ii) Micromanipulator method
Micromanipulators have been built, which permit one to pick out a single cell from a mixed culture. This instrument is used in conjunction with a microscope to pick a single cell (particularly bacterial cell) from a hanging drop preparation. The micro-manipulator has micrometer adjustments by means of which its micropipette can be moved right and left, forward, and backward, and up and down. A series of hanging drops of a diluted culture are placed on a special sterile coverslip by a micropipette.
Now a hanging drop is searched, which contains only a single microorganism cell. This cell is drawn into the micropipette by gentle suction and then transferred to a large drop of sterile medium on another sterile coverslip. When the number of cells increases in that drop as a result of multiplication, the drop is transferred to a culture tube having suitable medium. This yields a pure culture of the required microorganism.
The advantages of this method are that one can be reasonably sure that the cultures come from a single cell and one can obtain strains with in the species. The disadvantages are that the equipment is expensive, its manipulation is very tedious, and it requires a skilled operator. This is the reason why this method is reserved for use in highly specialized studies.
2. Enrichment Culture Method
Generally, it is used to isolate those microorganisms, which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixed culture. The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation. The medium of known composition and specific condition of incubation favors the growth of desired microorganisms but, is unsuitable for the growth of other types of microorganisms.
Proof of Purity of Cultures
Assuming that one has isolated a pure culture, how does one establish that it is pure? A pure culture is one in which the cells are all of one kind, i.e., demonstrate "likeness". Hence, the proof of purity of cultures consists of demonstrating the "likeness" of microorganisms in the culture. It is based on certain criteria as follows:
1.The microorganisms look alike microscopically and stain in the same fashion.
2. When plated, all the colonies formed look alike.
3. Streaks, stabs, etc. are uniform.
4. Several isolated colonies perform identically, i.e., ferment the same sugars, and so on.
Capillary method for obtaining a single microbial cell
Maintenance and Preservation of Pure Cultures
Once a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the microorganism by keeping the pure cultures free from contamination. Normally in laboratories, the pure cultures are transferred periodically onto or into a fresh medium (subculturing) to allow continuous growth and viability of microorganisms. The transfer is always subject to aseptic conditions to avoid contamination.
Since repeated sub culturing is time consuming, it becomes difficult to maintain a large number of pure cultures successfully for a long time. In addition, there is a risk of genetic changes as well as contamination. Therefore, it is now being replaced by some modern methods that do not need frequent subculturing. These methods include refrigeration, paraffin method, cryopreservation, and lyophilization (freeze drying).
Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms. This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus their growth continues slowly, nutrients are utilized and waste products released in medium. This results in, finally, the death of the microbes after sometime.
This is a simple and most economical method of maintaining pure cultures of bacteria and fungi. In this method, sterile liquid paraffin in poured over the slant (slope) of culture and stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture is preserved for several years.
Cryopreservation (i.e., freezing in liquid nitrogen at -196°C) helps survival of pure cultures for long storage times. In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of stabilizing agents such as glycerol that prevent the formation of ice crystals and promote cell survival.
In this method, the culture is rapidly frozen at a very low temperature (-70°C) and then dehydrated by vacuum. Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped as a result, the microbes go into dormant state and retain viability for years. Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark at 4°C in refrigerators. Freeze-drying method is the most frequently used technique by culture collection centers.
Lab 3: Obtaining Pure Cultures from a Mixed Population - Biology
As mentioned in the previous lab, pure cultures are essential for microbiological studies. Obtaining a pure culture of bacteria is usually accomplished by spreading bacteria on the surface of a solid medium so that a single cell occupies an isolated portion of the agar surface. This single cell will go through repeated multiplication to produce a visible colony of similar cells, or clones. There are three methods commonly used to derive a pure culture:
- Spread plate &ndash the original culture is diluted serially and a small volume of the final dilution is spread on the surface of an agar plate.
- Pour plate &ndashthe original culture is diluted serially and a small volume of the final dilution is added to molten agar which is poured over an agar plate and allowed to harden. Colonies develop sub-surface.
- Streak plate &ndash the original culture is directly diluted across an agar surface using and inoculating loop. This is a simple & rapid method.
All of these methods dilute or &ldquothin out&rdquo a heavy population of bacteria across an agar surface. The spread plate technique was used in lab #5 to obtain isolated colonies. In this lab we will learn to streak a plate with a mixed culture containing more than one bacterial species. Since most bacterial samples encountered by a microbiologist (in the clinic, environment, industry, etc.) are mixed cultures, this is a very important microbiological technique. If this procedure is performed correctly, a number of isolated colonies will grow that will be a source of pure bacterial cultures.
Streak plate - Triplet Streak. There are many variations of the streaking technique, but in this lab we will use the triplet streak as described here. Be sure to note the diagrams and description of the technique on the following page before starting with your streak plate.
Lab 3: Obtaining Pure Cultures from a Mixed Population - Biology
The process of screening a pure culture by separating one type of microbes from a mixture is called isolation. A culture containing only one species of microbe is called pure culture. In a mixed culture, a particular species is present in small numbers in comparision to the numbers of others.
A single viable cell may be transferred on the culture medium to develop axenic pure culture by using micromanipulator which is used with a microscope for picking up a single colony from a mixed population.
The nutrient agar slide or culture medium containing plate is exposed to the atmosphere for few minutes. After incubation, small colonies appear on the surface of medium which may be transferred on a fresh medium aseptically to obtain pure culture. Such technique is called sub- culturing. When the transfer is from solid medium (agar) to liquid medium (broth), the term picking off is used. In such cases the colour of the colony, their size, shape, appearance, form, consistency and optional properties are recorded.
By Streak Plate Technique
In this method the tip of a fine structure wire loop called inoculation needle consists 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 broth. 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 case, the colony from solid medium is streaked on the surface of nutrient agar medium in a sterile petridish.
By Inoculating In Animals
If disease causing microbe is unable to grow on artificial culture media, the impure culture is injected into the susceptible animals such as guinea pigs or rabbits. These animals allow disease causing organism to grow while rest of the microorganism i.e. etiologic microbe can be isolated on pure form from blood or affected tissue.
This medium is prepared by adding any number of growth factors. This addition enhances the growth and recovery of the desired microorganism. The differential media by virtue of their ingredients distinguish organism growing together. Thus, in EMB (eosin methylene blue complex), Escherichia coli imparts metallic sheen due to precipitation of eosin- methylene blue complex while other enrich bacteria generally do not show metallic sheen. On MacConkey agar medium, E.coli colonies are brick red in colour due to fermentation of lactose. On the other hand, Salmonella typhi does not give this appearance due to non- fermenter of lactose. The other media are selective media such as those which of their special composition promote the growth of one organism and inhibit the growth of others. The minimal medium lack certain growth factors. The medium supports the growth of those microorganisms whose nutritional requirements do not exceed those of the corresponding wild type of strain. Such media are helpful when auxotrophs are required .
The other methods to isolate microorganisms are: a. by controlling physical environment (especially temperature and pH), b. by culturing highly diluted microbial suspension by pour plate method in which isolated population of microorganisms growing from a single isolate can then be easily separated. In pour plate method, the sample is mixed with known quantity of distilled water. The suspension is plated (1 ml) on a presterilized petri dish, spread the sample with the help of loop, and then pour the melted agar medium after cooling it, over the sample containing plate. Incubate the petridish in an incubator at optimum temperature. If counting is not possible , then sample dilutions are made.
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STREAK PLATE CULTURE TECHNIQUE FOR THE ISOLATION OF MICROORGANISM / BACTERIA IN PURE CULTURE
A variety of techniques has been developed for the isolation of microorganism, mainly the bacteria, from the specimen or from the cultures and Streak plate technique is among the most widely used technique to obtain discrete & Well developed colonies of microbes.
The Culture techniques are commonly used in the laboratory for various purposes for which they are intended. The indications for the culture of the organism is mainly done –
- To demonstrate the cultural characteristics of the bacteria (e.g. color, texture, size, elevation etc.).
- To isolate the bacteria in discrete colonies from the specimen containing more than 1 bacterium.
- For determining the Sensitivity and/or Resistance of bacterium towards the particular Drug/Antibiotics or Test substances.
- To obtain the sufficient growth of the bacterium for various biochemical and other tests.
- To estimate the viable counts of the bacteria in the specimen.
- To maintain the stock cultures.
- To transport or short-term storage of the specimen (e.g. stab culture).
Three types of techniques are commonly employed for the isolations of microorganism from the specimen which are as follows:
In this article, we’ll discuss the Streak Plate technique for the isolation of microorganism in the microbiology laboratory.
PRINCIPLE OF STREAK PLATE TECHNIQUE
The Streak Plate technique for the isolation of microorganism is the most practical method of obtaining discrete and well-developed colonies of the microbe in pure cultures.
It was originally developed by the two bacteriologists, Loeffler and Gaffkey in the lab of Robert Koch (Father of Bacteriology).
In Streak plate technique, a sterilized loop or transfer needle is dipped into the mixed culture of the specimen or a suitably diluted suspension of organisms or Broth cultures, which is then streaked on the surface of Sterile Media plates to make parallel, non-overlapping streaks which are then incubated at optimum temperatures for 24-48 hours usually (for bacteria) or for few weeks in case of fungi.
The Aim of this method is to obtain the discrete, well-developed colonies of the microorganism that are pure, i.e. the growth derived from the single bacteria cell/spore.
REQUIREMENTS FOR THE POUR PLATE TECHNIQUE
- 24 hours old nutrient broth culture of two or more bacteria (Mixed Culture) or Sample/Specimen.
- Nutrient Agar Medium
- Six 9 ml Sterile Water Blanks
- Sterile Petri plates
- Graduated pipette (1ml or 1000 ml)
- Bunsen burner or Spirit lamp
PROCEDURE OF SPREAD PLATE TECHNIQUE
⇒ Prepare the Nutrient agar medium (NAM) and label the sterile Petri plate with the Patient identification no. & Date.
⇒ Now, pour the prepared Nutrient Agar Medium into the Sterile Petri plates under strict aseptic conditions. Allow the Media plates to Solidify.
Usually, the Streak plate method is done by streaking the specimen directly onto the Agar media plates but in some cases when the there is the more dense population of the organism in the specimen is suspected that a serial dilution of the specimen is done and then the diluted specimen is streaked onto the solidified agar media plates.
Follow the dilution step if required otherwise directly follow the Streaking protocol (T streak method or Quadrant method)
Serial Dilutions of the Specimen / Sample
⇒ Label the 6 Sterile Water blanks (9ml sterile water in each tube) as number 1 to 6 with the help of Marker. Also, label the Sterile Petri plates as number 1 to 6. Pour the Agar media in the plates and Kept aside to Solidify.
⇒ Place the labeled tubes in the test tube rack. Mix well the 24 hours old broth culture to equally distribute the bacterial cells in the tube.
⇒ After mixing, Remove the Cotton plug and aseptically transfer the 1 ml of the bacterial suspension from the tube of culture to sterile water blank tube no. 1 using a graduated pipette.
⇒ Shake the tube no. 1 to mix well the content to uniformly distribute the bacterial cells. Transfer the 1 ml of this to the water blank tube no. 2 by using the graduated pipette.
Note: Use the separate sterile pipette each time to transfer the contents from one tube to another.
⇒ In this way, make serial dilutions till the six water blanks (no. 1 to no. 6).
Inoculating / Streaking the Specimen on Media plates
There are various methods or patterns of streaking have been developed for inoculating the specimen on the media plates for the isolation of microorganisms.
VARIOUS PATTERNS OF STREAKING
But three methods are commonly used in most of the microbiology laboratories for the isolation of microorganism in pure cultures which are as follows –
- Parallel Streaking method (Quadrant)
- Zigzag streaking method (Quadrant)
- T streak Method (Three sectors)
The parallel Streak Quadrant Method & Zigzag Streak Quadrant Method are the Quadrant methods whereas the T- Streak method is the three sector method.
The Parallel Streak Quadrant method is commonly used in the Labs for the Isolation of Microorganism. However, the employment of the streaking methods and pattern of streaking varies from lab to lab.
PATTERN OF STREAKING VARIES – EVERY LABORATORY SETS THEIR OWN SOPs
Parallel Streak Quadrant Method
⇒ Hold the Specimen tube or Broth culture tube of mixed culture or the Diluted Specimen tube in the left hand.
⇒ Now, sterilize the inoculating loop in the flame of the Bunsen burner or Spirit lamp, holding in the Right hand. Remove the cotton plug of the tube and immediately flame the mouth of the tube.
⇒ Insert the Sterilized inoculating loop into the broth culture tube and withdraw one loopful of culture.
⇒ Promptly, flame the mouth of the tube, replace the cotton plug and place the tube in the test tube rack.
⇒ Now, hold the solidified sterile media plate in your left hand at an angle of 60° and place the inoculum (the loop containing the droplet of the specimen) on the agar surface in area 1 (at the edge farthest from you).
⇒ Flame the inoculating loop and cool for 5-10 seconds or promptly by touching to the unused area of solidified agar medium in the media plate. Streak the inoculum from side to side in parallel lines across the surface area 1.
⇒ Remove the loop and close the inoculated media plate. Reflame and cool the loop and turn the media plate 90° anti-clockwise and touch the sterilized inoculating loop to a corner of the culture in area 1 and drag it several times across the agar in area 2, hitting the original streak a few times. Remember that the loop should never enter area 1 again.
⇒ Remove the loop and close the inoculated media plate. Reflame and cool the loop and turn the media plate 90° anti-clockwise. Now Streak the area 3 in the same manner as area 2, hitting the last area several times.
⇒ Reflame and Cool the loop and turn the Media plate to 90°. Touch the loop to the corner of the culture streak in Area 3 and streak the inoculum across the agar.
⇒ Finally, the last streak is made in a zigzag manner by touching the corner of the culture streak in area 4 and streaking it in a zigzag manner making a tail in the culture. Remember not to overlap this closing tail with any of the Area of Quadrant.
⇒ Replace the Lid of the Inoculated Media plate and sterilize the inoculating loop in the flame of Bunsen burner or spirit lamp.
⇒ Incubate all the plates at optimum temperature, usually at 37 °C, in an inverted position for 24-48 hours.
Examine the plates for the appearance of individual colonies growing throughout the quadrants in the agar medium. The colonies outside the quadrant are considered as contamination.
RESULTS OF STREAK PLATE CULTURE TECHNIQUE
Generally, a confluent growth will be observed where the initial streak was made, the growth is less dense away from the streak, and discrete colonies will be there in the last area and tail streak.
If the diluted specimen was used to inoculate the media plates, the colonies in the cultures media plates will shows the lesser and lesser no. of the colonies with the increase in dilution factor that means the highest no. of colonies will develop in Plate no. 1 and least no. of colonies in plate no. 6 which will be distributes more or less sparsely along the streak lines.
PARALLEL STREAKS – QUADRANT STREAK PLATE TECHNIQUE
Select the discrete and well-developed colony and note down the characteristics like Color, Shape, Size, Appearance, Elevation, and Pigmentation etc.
These colonies may be transferred i.e. sub-cultured to the fresh media plates by streaking or other culture techniques to obtain the pure culture of the bacterial cells for further study.
PRECAUTIONS TO BE TAKEN…..
⇒ The protocol should be followed under all aseptic conditions preferably in Laminar Air Flow (Safety cabinet) to avoid any contamination.
⇒ Accurately measure the quantity while preparing the serial dilutions of the specimen.
⇒ Allow the media plates to uniformly solidify. Do not inoculate the specimen on partially solidified media plates.
⇒ Avoid pressing the inoculating loop too firmly against the agar surface.
⇒ The lid of the Petri plate should not be lifted completely to avoid any external contamination.
⇒ Sterilize the inoculating loop properly after streaking in an area of the quadrant to avoid contamination and to get the discrete & well-developed colonies.
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Pour Plate Method
This method involves plating of diluted samples mixed with melted agar medium.
The main principal is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a through distribution of bacteria cells with in the medium.
Here, the mixed culture of bacteria lis diluted directlyin the tubes containing melted agar medium maintained in the liquid state at a temepature(means successive dilutions of the inoculum (serially diluting the original specimen) are added into sterile petriplate to which is poured melted and cooled) 42ºC - 45ºC agar medium and thoroughly mixed by rotating the plates which is then allowed to solidify.
The content of each tube are poured into seprate petri plate , allowed to solidify, and then incubate
After incubation, the plates are examined for the presence of individual colonies.
The pure colonies may be isolated and transferred into test tube culture media for making pure cultures.
This technique is employed to estimate the viable bacterial count in a suspension.
Significance of Flaming:
Flaming the loop : Holding the loop in the flame of the Bunsen burner kills all contaminating organisms, thus sterilizing the loop. The loop should glow red-hot for a few seconds. After flaming, make sure to slightly cool the loop before picking up organisms from the inoculum culture (the culture that is to be transferred.) When transferring a culture from a plate, cool the loop by touching on the very edge of agar. When transferring from a broth, the red-hot loop will make a sizzling noise as soon as you insert it into the culture. The loop will automatically cool once it makes contact with the broth culture, but wait a one or two seconds before removing the loopful of inoculum from the tube. (The hot loop may create aerosols when it touches the media containing microorganisms. It will cause some of the broth and bacteria to boil briefly, creating a bacteria-containing aerosol. This airborne bacteria have the chances of entering into the respiratory tract or into the body parts. If you hear a hissing sound when you place the heat sterilized loop into the broth culture indicates that the loop is not cooled sufficiently).
Flaming the Mouth of the Test Tube: Passing the mouth of a tube through the flame of a Bunsen burner creates a convection current which forces air out of the tube. This prevents airborne contaminants from entering the tube. The heat of the Bunsen burner also causes the air around your work area to rise, reducing the chance of airborne microorganisms contaminating your cultures.
Agar Slants: Cultures are often transferred to agar slants, in addition to broth tubes and agar plates. An agar slant is a test tube containing agar, in which the solid agar forms a slant in the test tube. When inoculating an agar slant, draw the loop containing the inoculum very lightly over the surface in a zigzag formation while being careful not to break the surface. A needle can be used instead of a loop to inoculate an agar slant by stabbing the needle containing the inoculum into the agar ( Fig 1).
Trying to study this mixed population is often difficult and in the tradition of the scientific method researchers dissect a system and study each piece in isolation. For microorganisms this means separating the organisms and getting them into pure culture. A pure culture is defined as a growth of microorganisms (a culture) that contains one cell type. It is essential in microbiology to be able to obtain and preserve pure cultures. Over 100 years ago, Robert Koch devised methods to achieve this goal and the methods he developed are essentially still used today. The protocols used to maintain pure cultures are a major part of aseptic technique and are the subject of this chapter.
The goals of aseptic technique are two-fold. The first objective is to obtain pure cultures and secondly to prevent cross-contamination. Microorganisms in culture must not escape into the environment, and microbes in the environment must not get into the cultures we are studying. It is essential that aseptic technique be understood and practiced correctly. Contaminated cultures are worthless for diagnosis or for doing research on, because it is unclear what microbe is performing any action that is being observed.
Aseptic methods commonly used are flame sterilization, tube transfer, streak plates, spread plates and pour plates. Flame sterilization is an easy method to insure sterile transfer of a culture from a source to a growth medium. Tube transfer is useful for moving inocula from one tube to another. Mechanical dilution by making streak plates is the preferred method for obtaining a pure culture of a microorganism. Finally, spread plates and pour plates are common methods for enumerating microorganisms and are sometimes useful for obtaining isolated colonies.
Genetics Proves Indian Population Mixture
Between 4,000 and 2,000 years ago, intermarriage in India was rampant. Figure by Thangaraj Kumarasamy
Scientists from Harvard Medical School and the CSIR-Centre for Cellular and Molecular Biology in Hyderabad, India, provide evidence that modern-day India is the result of recent population mixture among divergent demographic groups.
The findings, published August 8 in the American Journal of Human Genetics, describe how India transformed from a country where mixture between different populations was rampant to one where endogamy—that is, marrying within the local community and a key attribute of the caste system—became the norm.
“Only a few thousand years ago, the Indian population structure was vastly different from today,” said co–senior author David Reich, professor of genetics at Harvard Medical School. “The caste system has been around for a long time, but not forever.”
In 2009, Reich and colleagues published a paper based on an analysis of 25 different Indian population groups. The paper described how all populations in India show evidence of a genetic mixture of two ancestral groups: Ancestral North Indians (ANI), who are related to Central Asians, Middle Easterners, Caucasians, and Europeans and Ancestral South Indians (ASI), who are primarily from the subcontinent.
However, the researchers wanted to glean clearer data as to when in history such admixture occurred. For this, the international research team broadened their study pool from 25 to 73 Indian groups.
The researchers took advantage of the fact that the genomes of Indian people are a mosaic of chromosomal segments of ANI and ASI descent. Originally when the ANI and ASI populations mixed, these segments would have been extremely long, extending the entire lengths of chromosomes. However, after mixture these segments would have broken up at one or two places per chromosome, per generation, recombining the maternal and paternal genetic material that occurs during the production of egg and sperm.
By measuring the lengths of the segments of ANI and ASI ancestry in Indian genomes, the authors were thus able to obtain precise estimates of the age of population mixture, which they infer varied about 1,900 to 4,200 years, depending on the population analyzed.
While the findings show that no groups in India are free of such mixture, the researchers did identify a geographic element. “Groups in the north tend to have more recent dates and southern groups have older dates,” said co-first author Priya Moorjani, a graduate student in Reich’s lab at Harvard Medical School. “This is likely because the northern groups have multiple mixtures.”
“This genetic datatells us a three-part cultural and historical story,” said Reich, who is also an associate member of the Broad Institute. “Prior to about 4000 years ago there was no mixture. After that, widespread mixture affected almost every group in India, even the most isolated tribal groups. And finally, endogamy set in and froze everything in place.”
“The fact that every population in India evolved from randomly mixed populations suggests that social classifications like the caste system are not likely to have existed in the same way before the mixture,” said co–senior author Lalji Singh, currently of Banaras Hindu University, in Varanasi, India, and formerly of the CSIR-Centre for Cellular and Molecular Biology. “Thus, the present-day structure of the caste system came into being only relatively recently in Indian history.”*
But once established, the caste system became genetically effective, the researchers observed. Mixture across groups became very rare.
“An important consequence of these results is that the high incidence of genetic and population-specific diseases that is characteristic of present-day India is likely to have increased only in the last few thousand years when groups in India started following strict endogamous marriage,” said co–first author Kumarasamy Thangaraj, of the CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India.**
Mohan Rao, Director, CSIR-CCMB said, “CCMB's continuing efforts over a decade on this field had helped in understanding the complexity of Indian population history and social structure, such as caste systems.”
This study was funded by the NIH (GM100233) NSF (HOMINID grant 1032255) a UKIERI Major Award (RG-4772) the Network Project (GENESIS: BSC0121) fund from the Council of Scientific and Industrial Research, Government of India a Bhatnagar Fellowship grant from the Council of Scientific and Industrial Research of the Government of India and a J.C. Bose Fellowship from Department of Science and Technology, Government of India.
*, ** Quotes adapted from American Journal of Human Genetics news release.