A Pure Culture Contains Only

Lab iii: Obtaining Pure Cultures from a Mixed Population

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DISCUSSION

Equally stated in Lab 2, microorganisms exist in nature as mixed populations. Nonetheless, to study microorganisms in the laboratory we must have them in the form of a pure civilization, that is, ane in which all organisms are descendants of the same organism. Two major steps are involved in obtaining pure cultures from a mixed population:

First, the mixture must exist diluted until the various private microorganisms become separated far enough apart on an agar surface that later on incubation they class visible colonies isolated from the colonies of other microorganisms. This plate is called an isolation plate.
And then, an isolated colony tin can exist aseptically "picked off" the isolation plate (Effigy i) and transferred to new sterile medium (see Fig. 3). Later on incubation, all organisms in the new culture will exist descendants of the same organism, that is, a pure culture.

pick off colony_final.jpg

Figure 1: Picking a Single Colony 0ff of a Petri Plate in order to Obtain a Pure Civilization Before removing bacteria from the petri plate, commencement cool the loop by sticking information technology into the agar away from any growth.

A. STREAK PLATE METHOD OF ISOLATION

The most common manner of separating bacterial cells on the agar surface to obtain isolated colonies is the streak plate method nosotros 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 than and more bacteria are rubbed off until individual separated organisms are deposited on the agar. Afterward incubation, the area at the beginning of the streak design will show confluent growth while the area near the end of the pattern should prove discrete colonies (see Fig. 2A and Fig. 2B).

B. THE Cascade PLATE AND SPIN PLATE METHODS OF ISOLATION

Another method of separating bacteria is the pour plate method. With the cascade plate method, the bacteria are mixed with melted agar until evenly distributed and separated throughout the liquid. The melted agar is and so poured into an empty plate and allowed to solidify. After incubation, discrete bacterial colonies can and 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. Minor samples of the diluted bacteria are so pipetted onto the surface of agar plates. A sterile, bent-glass rod is then used to spread the bacteria evenly over the unabridged agar surface (see Fig. 4) in gild to meet isolated colonies (come across Fig. 5). In Lab four we volition utilize this technique as part of the plate count method of enumerating bacteria.

C. USE OF SPECIALIZED MEDIA

To supplement mechanical techniques of isolation such every bit the streak plate method, many special-purpose media are bachelor to the microbiologist to assist in the isolation and identification of specific microorganisms. These special purpose media autumn into 4 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 ane group of organisms while permitting the growth of another. For case, Columbia CNA agar has the antibiotics colistin and nalidixic acrid added which inhibit the growth of Gram-negative bacteria but non 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 appreciable color change in the medium when a particular chemical reaction occurs. They are useful in differentiating bacteria according to some biochemical feature. In other words, they bespeak whether or not a certain organism tin 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 yous wish to culture is present in relatively modest numbers compared to the other organisms growing in the mixture.

four. Combination selective and differential media: A combination selective and differential medium permits the growth of i group of organisms while inhibiting the growth of another. In addition, it differentiates those organisms that abound based on whether they can conduct out particular chemical reactions.

For example, MacConkey agar (meet Fig. six) 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 equally bile salts that inhibit the growth of near Gram-positive leaner merely practice not affect the growth of nearly Gram-negatives. If the Gram-negative bacterium ferments the sugar lactose in the medium, the acrid end products lower the pH of the medium. The neutral cherry in the agar turns ruddy in colour once the pH drops below 6.viii. As the pH drops, the neutral red is absorbed by the leaner, causing the colonies to appear bright pink to red.

Results are interpreted as follows:

Potent fementation of lactose with loftier levels of acid production by the leaner causes the colonies and confluent growth to announced bright pink to red. The resulting acid, at high plenty 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 (come across Fig. 7).
Weak fermentation of lactose by the leaner causes the colonies and confluent growth to appear pink to cherry, simply without the atmospheric precipitation of bile salts in that location is no pinkish 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 equally follows:

Escherichia coli : colonies and confluent growth appear bright pink to cherry and surrounded past 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 only are not surrounded by a pink precipitate (cloudiness) in the agar surrounding the growth (run across Fig. 8).

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

There are literally hundreds of special-purpose media available to the microbiologist. Today we volition 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 hereafter labs, such as 12 - 16, which deal with the isolation and identification of pathogenic bacteria, nosotros will use many additional special-purpose media.

MEDIA

One plate of each of the post-obit 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 one of the following Gram-negative leaner:

Possible Gram-positive leaner:
- Micrococcus luteus. A Gram-positive coccus with a tetrad or a sarcina system; produces circular, convex colonies with a yellow, h2o-insoluble pigment on Trypticase Soy agar.
  - Micrococcus luteus growing on TSA
  - Shut up of Micrococcus luteus growing on TSA
- Staphylococcus epidermidis. A Gram-positive coccus with a staphylococcus arrangement; produces circular, convex, not-pigmented colonies on Trypticase Soy agar.
  - Staphylococcus epidermidis growing on TSA
  - Shut up of Staphylococcus epidermidis growing on TSA
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, perhaps mucoid colonies on Trypticase Soy agar.
  - Enterobacter aerogenes growing on TSA

During the adjacent 3 labs you will effort to obtain pure cultures of each organism in your mixture and determine which 2 bacteria you take. Today yous will endeavor to carve up the bacteria in the mixture in order to obtain isolated colonies; next lab you lot will place the 2 bacteria in your mixture and pick off single isolated colonies of each of the 2 bacteria in gild to get a pure civilisation of each. The post-obit lab you volition set up microscopy slides of each of the two pure cultures to make up one's mind if they are indeed pure.

Procedure (to exist done in pairs)

1. On the bottom of each of the three petri plate yous are using today, divide the plate into thirds with your wax marker and label equally shown below. This will guide your streaking.

two . Although Trypticase Soy agar (TSA), which grows both Gram-positive and Gram-negative bacteria, is non normally used as an isolation medium, we will try to obtain isolated colonies of the two organisms in your mixture by using strictly mechanical methods. Frequently, still, one bacterium overgrows another in a mixture and by the fourth dimension you spread out the more abundantant organism enough to become isolated colonies, the one in smaller numbers is no longer on the loop so yous may not meet single colonies of each on the TSA next time.

Streak your mixture on a plate of Trypticase Soy agar using 1 of the 2 streaking patterns illustrated in Lab ii, Fig. 4 and Fig. 5. agar

3 . Streak the same mixture for isolation (meet Fig. 4 and 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 . Streak the aforementioned mixture for isolation (run into Fig. 4 and Fig. v) on a plate of MacConkey agar (selective for Gram-negative bacteria and differential for certain members of the bacterial family unit Enterobacteriaceae).

Escherichia coli growing on MacConkey agar.
Enterobacter aerogenes growing on MacConkey agar.

v . 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.

RESULTS

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
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 selection off a single isolated colony of each of the 2 bacteria from your original mixture that you lot have but identified and aseptically transfer them to divide plates of Trypticase Soy agar (see Fig. three). Recall to streak the plate for isolation equally y'all learned in labs ii and 3.

b. When picking off single colonies, remove the top portion of the colony without touching the agar surface itself to avert picking upwards any inhibited bacteria from the surface of the agar. Make certain you write the proper noun of the bacterium (genus and species) you are growing on that TSA plate.

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

Performance OBJECTIVES FOR LAB iii

Afterward 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 take to eventually obtain pure cultures of each organism.

ii. Define: selective medium, differential medium, enrichment medium, and combination selective-differential medium.

3. Country the usefulness of Columbia CNA agar and MacConkey agar.

4. 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

1. 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 Canton, CATONSVILLE CAMPUS)