Pure Cultures - Methods of Isolating Pure Cultures With Diagrams

By Momina Shehzadi | BS Zoology Graduate, Pakistan

In this article, we will learn Pure Cultures and Methods of Isolating Pure Cultures - Streak Plate, Pour Plate, Spread Plate and Micromanipulator - in simple words with diagrams. This topic is from Honey General Microbiology Chapter 6, very important for BS Zoology and Microbiology students.

PURE CULTURES - Definition

When bacteria or other micro-organisms grow on a laboratory medium, they are referred to as a culture. A pure culture consists of a population of cells which are derived from a single cell. OR Culture consisting of a population of single species is known as a pure culture.

In simple words, if only one kind of organism grows, it is pure culture.

Method of Isolation of pure culture:

A variety of techniques have been developed whereby isolation into pure culture can be accomplished. Each technique has certain advantages and limitations, and there is no one method that can be used for all bacteria.

1) The Streak-Plate Technique - Most Important

By means of a transfer loop, a portion of the mixed culture is placed on the surface of an agar medium and streaked across the surface. This manipulation 'thins out' the bacteria on the agar surface so that some individual bacteria are separated from each other. When streaking is properly performed, the bacterial cells will be sufficiently far apart in some areas of the plate to ensure that the colony developing from one cell will not merge with that growing from another. A modification known as the roll-tube technique that is used for the isolation of stringent anaerobes.

Procedure 

(a) Flaming the loop: Entire length of wire heated until red hot - further heating unnecessary. Avoid the part of the flame with unburnt gas. Loop - allow to cool for about 10 seconds before use, e.g. by tapping handle against the base of the bunsen burner with loop in the air, or by touching onto the agar before use.

(b) Cooling the loop

(c) Streaking the plate:Collect a loopful of the sample if liquid. Touch the sample with the loop if solid. Raise the lid of the petri dish only as far as necessary and lightly spread the sample as shown, without damaging the surface of the agar. Keep the loop flat and hold the handle at the balance point near the centre. Each line represents one stroke with the wire loop. Numbers show the sequence of strokes (1 to 24). Flame the loop and cool between each set of six lines. Flame again at the end. Label the base of the plate. Incubate upside down to prevent any condensation falling onto the cultures.

- Labelling the plate - date, initials, contents. If you use mirror-writing you will be able to read it through the lid.

(d) Final appearance after incubation: Individual colonies formed from individual bacteria. The process can be repeated using one of these colonies to check purity if necessary. A pure culture can be obtained in this way.

Fig. Streak-plate culture showing areas of isolated colonial growth. Note that where the colonies are sparse they are larger than when crowded together. (Courtesy of Naval Biological Laboratory.)

Fig. Roll-tube method for isolating stringent anaerobes: (A) Stoppered anaerobic culture tube whose inner walls have been coated with a prereduced agar medium. The tube contains an atmosphere of oxygen-free N2. (B) When the stopper is removed the tube is kept anaerobic by continuously flushing it with oxygen-free CO2 from a gas cannula. Inoculation is done with a transfer loop held against the agar surface as the tube is being rotated by a motor. By starting at the bottom and drawing the loop gradually upward, the inoculum becomes "thinned" to the point where well-isolated colonies can develop. (C) After inoculation the tube is restoppered and incubated.

Important Concept about Clone:The assumption is often made that a colony is derived from a single cell and, therefore, that the colony is a clone. However, this is not necessarily true. With species in which the cells form a characteristic grouping during cell division (for example, clumps of staphylococci or chains of streptococci), the colony develop from a group of cells rather than from a single cell. Although not a clone, such a colony is nevertheless a pure culture if it contains only one kind of organism.

One should recognize that subculturing a colony from a single streak plate does not automatically assure purity. The colony may have been derived from two or more different kinds of bacteria. For example, when we attempt to isolate slime- or chain-producing bacteria, contaminants may be found to have adhered to the slime or to have been enmeshed in the network of chains, thereby resulting in impure colonies. The use of selective media can also lead to impure colonies. Although the growth of contaminants is inhibited on selective media, low numbers of viable cells may still be present, and such cells can be subcultured along with a colony. For these reasons, it is advisable to streak a culture several times in succession, preferably on nonselective media, in order to ensure purity.

2) The Pour-Plate and Spread-Plate Techniques

Unlike the streak-plate technique, the pour-plate and the spread-plate techniques is performed in a quantitative manner to determine the number of bacteria (of a particular type) present in a specimen.

In both of these methods the mixed culture is first diluted to provide only a few cells per milliliter before being used to inoculate media. Since the number of bacteria in the specimen is not known beforehand, a series of dilutions is made so that at least one of the dilutions will contain a suitably sparse concentration of cells.

i) Pour-plate method 

In the pour-plate method the mixed culture is diluted directly in tubes of liquid (cooled) agar medium. The medium is maintained in a liquid state at a temperature of 45°C to allow thorough distribution of the inoculum. The inoculated medium is dispensed into Petri dishes, allowed to solidify, and then incubated. A series of agar plates showing decreasing numbers of colonies resulting from the dilution procedure in the pour-plate technique.

Fig. Pour-plate technique: Step 1: One loopful of original bacterial suspension is transferred to tube A (liquid, cooled agar medium). Tube A is rolled between the hands to effect thorough mixing of inoculum. Similar transfers are made from A to B to C. Step 2: Contents of each tube are poured into separate Petri dishes. Step 3: After incubation, plates are examined for the one which contains well-separated colonies. From this plate, pure cultures of bacteria can be isolated by transferring a portion of a colony to a tube of sterile medium.

Disadvantages of Pour-plate:

i) Some of the organisms are trapped beneath the surface of the medium when it gels. Therefore both surface and subsurface colonies develop. The subsurface colonies can be transferred to fresh media only by first digging them out of the agar with a sterile instrument.

ii) Another disadvantage is that the organisms being isolated must be able to withstand temporary exposure to the 45°C temperature of the liquid agar medium.

Procedure for pouring a plate 

Allow liquid nutrient agar to cool to about 50°C (just cool enough for comfortable handling) after removal from autoclave. Unscrew cap of bottle with little finger as shown. It can be held by the little finger during the procedure to avoid placing it on the bench where it could become contaminated. 'Flame' mouth of bottle for a few seconds. This is done to produce an upward flow of air from the bottle so that any organisms in the area will not fall into the bottle. Pour the molten agar (about 15-20cm³) slowly into the base of the sterile petri dish, lifting the lid only as much as necessary. Do not spill agar on the edges of the dish (if so, start again). Replace the lid and allow the agar to set. Plates are dried with base resting on lid as shown. If the plate is to be used for 'streaking' or 'spreading' the surface of the agar and lid can be dried once the agar is set. They are best dried upside down in an incubator at about 37°C, and arranged as shown to minimise the risk of contamination.

ii) Spread-plate method

In the spread-plate method the mixed culture is not diluted in the culture medium; instead it is diluted in a series of tubes containing a sterile liquid, usually water or physiological saline. A sample is removed from each tube, placed onto the surface of an agar plate. It is spread evenly over the surface by means of a sterile bent glass rod. On at least one plate of the series the bacteria will be in numbers sufficiently low as to allow the development of well-separated colonies. In contrast to the pour-plate technique, only surface colonies develop. Moreover, the organisms are not required to withstand the temperature of liquid agar.

Fig. Spread culture (Page 144): (a) Stand the spreader in a small beaker of 70% alcohol. (b) Transfer a known, small volume (up to 0.5cm³) of cell suspension to the surface of the nutrient agar in a petri dish. (c) Remove the spreader from the alcohol and allow excess alcohol to drain from it. Ignite the alcohol by passing the spreader through a bunsen flame. (d) Push the liquid over the surface of the agar using the spreader. Rotate the plate at the same time to ensure an even coverage. Label the base of the plate and incubate. (e) Final appearance after incubation: Count the number of colonies. This corresponds to the number of bacteria originally inoculated.

3) Micromanipulator Techniques

A device called the micromanipulator is used in conjunction with a microscope to pick a single bacterial cell from a mixed culture. The micromanipulator permits the operator to control the movements of a micropipette or a microprobe (a fine needle) so that a single cell can be isolated. This technique requires a skilled operator. It is reserved for studies in which a clone must be obtained clearly.

Conclusion

This is complete Chapter 6 from Honey Microbiology. For exam, you must remember Streak plate diagram (1-24 strokes) and Pour plate 3 steps. This is 10 marks question.

FAQs for Students - Pure Cultures

Q1: What is pure culture in microbiology? Simple definition.

Ans: Pure culture is a culture which contains only one species of organism. It is derived from a single cell. For example, if only _E. coli_ grows on agar plate, it is pure culture. It is needed to study morphology and biochemical properties of bacteria.

Q2: What is difference between pure culture and mixed culture?

Ans: Pure culture = Single species (Akela). Mixed culture = Many species growing together. In lab we need pure culture for correct identification, mixed culture gives wrong results.

Q3: Which is the most common method for isolation of pure culture?

Ans: Streak-plate technique is the most common and easiest method. It is used in every microbiology lab. It works on principle of thinning by streaking with transfer loop.

Q4: What is streak plate method in simple words?

Ans: In streak plate, we take a loopful of mixed bacteria and streak line by line (1 to 24 strokes) on agar plate. After incubation, well-isolated colonies appear at the end of streaks. Flame the loop after every 6 strokes.

Q5: What is Roll-tube technique?

Ans: Roll-tube is modification of streak-plate for stringent anaerobes (bacteria which die in oxygen). Tube walls are coated with agar, tube contains N2 gas, and inoculation is done while tube rotates by motor with CO2 flushing.

Q6: What is Pour-plate method and at what temperature agar is kept?

Ans: In pour-plate, mixed culture is diluted in tubes of liquid agar at 45°C. Then agar is poured into plates. Both surface and subsurface colonies develop. Its disadvantage is that bacteria must tolerate 45°C and subsurface colonies are hard to pick.

Q7: What is Spread-plate method?

Ans: In spread-plate, bacteria is diluted in water/saline, not in agar. A small volume is placed on agar surface and spread evenly with sterile bent glass rod (hockey stick). Only surface colonies develop. It is used for counting bacteria (quantitative method).

Q 8: Pour plate vs Spread plate - main difference?

Ans: Pour = Bacteria INSIDE agar (Andar), colonies both surface + subsurface, need 45°C tolerance. Spread = Bacteria ON TOP of agar (Upar), only surface colonies, no heat damage. Spread is better for counting.

Q 9: What is micromanipulator technique?

Ans: Micromanipulator is a device used with microscope to pick a single bacterial cell with micropipette or microprobe. It is very difficult and needs expert operator. It is used when we need a true clone from single cell.

Q 10: Is a colony always a clone?

Ans: No. Colony from single cell is clone, but in bacteria like staphylococci (clumps) or streptococci (chains), colony develops from group of cells. But it is still pure culture if only one species present.

Q11: How to ensure purity of culture?

Ans: Subculturing from single streak plate does not assure purity. Slime or chain-producing bacteria can carry contaminants. So it is advisable to streak culture several times in succession, preferably on nonselective media.

Q12: Why pure culture is important for exam (5 marks)?

Ans: Pure culture is important because: 1) To study shape, size, staining of one bacterium, 2) To test which antibiotic works, 3) To study biochemical reactions, 4) To prepare vaccines, 5) To avoid contamination.

Reference:Concept taken from Honey General Microbiology, explained with original examples and diagrams for exam preparation.

Also Read: Size, Shapes and Arrangements of Bacterial Cells

Written by Momina Shehzadi, BS Zoology Graduate from Pakistan.