Can a Pure Culture Be Prepared From a Mixed Broth

Lab 3: Obtaining Pure Cultures from a Mixed Population

1. Concluding updated

2. Save as PDF

Page ID

3442

DISCUSSION

As stated in Lab 2, microorganisms exist in nature as mixed populations. However, to report microorganisms in the laboratory nosotros must have them in the form of a pure civilisation, that is, one in which all organisms are descendants of the same organism. Ii major steps are involved in obtaining pure cultures from a mixed population:

1. First, the mixture must be diluted until the various individual microorganisms go separated far enough apart on an agar surface that after incubation they course visible colonies isolated from the colonies of other microorganisms. This plate is called an isolation plate.
2. Then, an isolated colony tin can exist 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 gild to Obtain a Pure Civilisation 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 near common fashion 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 past mechanical means. As the loop is streaked across the agar surface, more and more than bacteria are rubbed off until individual separated organisms are deposited on the agar. After incubation, the area at the beginning of the streak design will show confluent growth while the area almost 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

Some other method of separating bacteria is the pour plate method. With the pour plate method, the leaner 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 tin then be plant 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. Modest samples of the diluted bacteria are then pipetted onto the surface of agar plates. A sterile, aptitude-glass rod is so used to spread the bacteria evenly over the entire agar surface (see Fig. four) in social club to see isolated colonies (run into Fig. v). In Lab 4 we will use this technique as part of the plate count method of enumerating bacteria.

C. Apply OF SPECIALIZED MEDIA

To supplement mechanical techniques of isolation such as the streak plate method, many special-purpose media are bachelor 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 acrid 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 item chemic reaction occurs. They are useful in differentiating bacteria according to some biochemical characteristic. In other words, they indicate whether or not a sure organism can carry out a specific biochemical reaction during its normal metabolism. Many such media will exist 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 improver, it differentiates those organisms that grow based on whether they can carry out particular chemic reactions.

For example, MacConkey agar (run across Fig. half-dozen) 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 leaner but exercise non touch the growth of most Gram-negatives. If the Gram-negative bacterium ferments the sugar lactose in the medium, the acid stop products lower the pH of the medium. The neutral red in the agar turns red in color once the pH drops beneath 6.8. As the pH drops, the neutral scarlet is absorbed by the leaner, causing the colonies to appear bright pink to red.

Results are interpreted as follows:

• Strong fementation of lactose with loftier levels of acid production by the bacteria causes the colonies and confluent growth to appear vivid pinkish 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 announced in the agar surrounding the growth (see Fig. seven).
• Weak fermentation of lactose by the bacteria causes the colonies and confluent growth to appear pink to cerise, but without the precipitation of bile salts there is no pink precipitate (cloudiness) in the agar surrounding the growth (see Fig. viii).
• If the bacteria do non 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 announced brilliant pink to crimson 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 ruby but are not surrounded by a pink precipitate (cloudiness) in the agar surrounding the growth (run into Fig. eight).

Salmonella, Serratia, Proteus, and Shigella: colorless colonies; agar relatively transparent (encounter Fig. 9).

In that location are literally hundreds of special-purpose media available to the microbiologist. Today nosotros will combine both a mechanical isolation technique (the streak plate) with selective and selective-differential media to obtain pure cultures from a mixture of leaner. In futurity labs, such every bit 12 - sixteen, which deal with the isolation and identification of pathogenic bacteria, we volition apply many additional special-purpose media.

MEDIA

One plate of each of the following media: Trypticase Soy agar, Columbia CNA agar, and MacConkey agar.

ORGANISMS

A broth culture containing a mixture of one of the post-obit Gram-positive bacteria and ane 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 upwards of Micrococcus luteus growing on TSA
  • Staphylococcus epidermidis. A Gram-positive coccus with a staphylococcus organization; produces round, convex, not-pigmented colonies on Trypticase Soy agar.
    • Staphylococcus epidermidis growing on TSA
    • Close up of Staphylococcus epidermidis growing on TSA
• Possible Gram-negative bacteria:
  • Escherichia coli. A Gram-negative bacillus; produces irregular, raised, not-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

During the adjacent three labs you will attempt to obtain pure cultures of each organism in your mixture and determine which two bacteria you lot accept. Today y'all will endeavor to separate the leaner in the mixture in club to obtain isolated colonies; side by side lab y'all will identify the ii bacteria in your mixture and pick off single isolated colonies of each of the two bacteria in order to become a pure civilization of each. The following lab you will prepare microscopy slides of each of the ii 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 yous are using today, separate the plate into thirds with your wax mark and label as shown beneath. This volition guide your streaking.

ii . Although Trypticase Soy agar (TSA), which grows both Gram-positive and Gram-negative bacteria, is not ordinarily used equally an isolation medium, nosotros will attempt to obtain isolated colonies of the two organisms in your mixture by using strictly mechanical methods. Often, yet, one bacterium overgrows some other in a mixture and past the fourth dimension you spread out the more abundantant organism enough to get isolated colonies, the one in smaller numbers is no longer on the loop so yous may not see single colonies of each on the TSA adjacent fourth dimension.

Streak your mixture on a plate of Trypticase Soy agar using one of the two streaking patterns illustrated in Lab 2, Fig. iv and Fig. 5. agar

3 . Streak the same mixture for isolation (see Fig. 4 and Fig. 5) on a plate of Columbia CNA agar (selective for Gram-positive leaner).

• Micrococcus luteus growing on Columbia CNA agar.
• Staphylococcus epidermidis growing on Columbia CNA agar.

4 . Streak the same mixture for isolation (see Fig. iv and Fig. five) on a plate of MacConkey agar (selective for Gram-negative leaner 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 respective to your lab section until the next lab flow.

RESULTS

1. Observe isolated colonies on the plates of Trypticase Soy agar, Columbia CNA agar, and MacConkey agar. Tape your observations and conclusions.

Trypticase Soy agar
Observations
Conclusions
Columbia CNA agar
Observations
Conclusions
MacConkey agar
Observations
Conclusions

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 leaner from your original mixture that you have only identified and aseptically transfer them to separate plates of Trypticase Soy agar (see Fig. 3). Remember to streak the plate for isolation as you learned in labs 2 and iii.

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 certain you write the name of the bacterium (genus and species) you are growing on that TSA plate.

c. I ncubate the plates upside down in your petri plate holder at 37°C until the next lab menstruum. These volition exist your pure cultures for Lab 5 (Directly and Indirect stains).

Functioning OBJECTIVES FOR LAB 3

Afterwards completing this lab, the student will be able to complete the following objectives:

DISCUSSION

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 have 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.

iv. Describe how each of the post-obit would appear when grown on MacConkey agar:

a. Escherichia coli
b. Enterobacter aerogenes
c. Salmonella

PROCEDURE

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.

RESULTS

i. When given a plate of Columbia CNA agar or MacConkey agar showing discrete colonies, correctly interpret the results.

Cocky-QUIZ

Contributors and Attributions

• Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)

jacquesthersemeaten.blogspot.com

Source: https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_II/Lab_03%3A_Obtaining_Pure_Cultures_from_a_Mixed_Population

Bagikan Artikel ini