Essay: Microbiological Techniques: Isolating Pure Culture Growth from Mixed Populations



Contamination and infection caused by microorganisms are a key concern in the scientific and medical community.1 It is important for one to become familiar with the abundance of microorganisms and the practice of aseptic technique. This experiment examined the use of various basic microbiological and aseptic techniques to derive pure culture growth from mixed colonies and the ubiquity of microorganisms.2 Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were isolated from a mixed population and pure colonies were derived of each bacterium strain.2 The use of various media allowed for one to study the ideal growing conditions of bacteria, at optimal incubation temperature and access to nutrients.2 The media were compared based on effectiveness for different experimental objectives such as isolation versus propagating large colonies.2 Control groups involving sterile inoculating tools were found to contain no bacterial growth. This is significant as it confirmed the success of the aseptic techniques in place throughout the experiment. Furthermore, media exposed to surfaces and the laboratory room showed extensive bacterial growth. This result exemplified the vast existence of microorganisms in various environments. These results have important implications for sterility and colony growth in the scientific and medical field.3

Introduction

  Microbiology is the study of small organisms that require a microscope to be seen, such as bacteria, protists, and viruses.2 Microorganisms are found in a large number of diverse environments, including those that are harmful to people and animals. Pathogens are microbes that cause diseases.4 Scientists combat these pathogens with vaccines, antibodies, and aseptic techniques.4 In this laboratory experiment, aseptic techniques were practiced to observe whether there was contamination present in the media cultures, with the goal of achieving effective controls with no growth.2 On the other hand, microbes can be beneficial to humans for purposes spanning from the food industry to medical benefits.4  This laboratory involved the use of various microbiological techniques to isolate pure cultures from mixed populations with the purpose of growing a new, pure culture from a previously mixed one.2 The presence of bacteria is almost limitless, as can be seen by simply sampling any surface and swabbing a nutrient agar media. The result will be bacteria growth, as its presence is abundant and diverse.

   The ubiquity of microorganisms refers to the notion that they can be found in an abundance of environments, including the air, in plants, and even in the laboratory setting.1 Generally, microorganisms contribute a byproduct to their environment. A common example is Cyanobacteria, capable of nitrogen fixing, making nitrates available for plants to use.5 Generally, bacteria are structurally and metabolically diverse, existing in symbiotic relationships with their surroundings.5 Nevertheless, bacteria can be harmful, causing disease and contaminating samples in the laboratory.3 For this reason, measures are in place to avoid contamination when working with cultures.

   Aseptic technique is a broad term referring to conditions that must be upheld in a laboratory setting to avoid contamination- commonly through sterility.2 Contaminants can alter test results, cause disease, and spoil costly results. Working in an aseptic environment involves but is not limited to washing hands, wearing proper laboratory attire, working on cultures under a biological safety cabinet, disinfecting laboratory benches, and using sterilized equipment.2 Sterilized inoculating tools such as pipettes and swabs are used to transfer microorganisms from samples to culture media.2 As exemplified in this laboratory, bacteria are everywhere, including the air. Thus, it is essential to be aware of the exposure to air that sterilized tools experience and limit it to avoid contamination. Inoculating tools such as loops tend to be reused for transfers of bacteria. Accordingly, great care must be taken when sterilizing them. This is often done with a heat source to eliminate any bacteria present. Aseptic techniques are used under the impression that bacteria are everywhere including the laboratory setting and contamination must be limited.3

  Nutrients and an ideal growth environment must be available for a culture of microorganisms to survive and grow. In a laboratory setting, this environment is provided in the form of culture media. Three common forms of culture media exist, each with its own advantages. The point of differentiation is the presence of a gelling agent.2 Broth media do not have gelling agents such as agar, however they do have nutrients available.6 These media are most often used for the propagation of a large population of microorganisms in fermentation studies within test tubes or flasks.6 In contrast, semisolid media and solid media contain a solidifying agent such as agar.2 Semisolid media are often contained in test tubes, as it is easier to observe the motility of microorganisms and promote anaerobic growth.2 Lastly, solid media contain a higher concentration of agar, allowing microorganisms to grow in colonies or streaks.6 This is useful for the isolation and storage of microorganisms, as well as observation purposes.2 It is important to note that all nutrient-containing media are sterile prior to inoculation with bacteria.

   In nature, bacteria tend to be found in mixed populations of species, or mixed cultures because various types of bacteria tend to form symbiotic or mutualistic relationships with one another.5 This makes it difficult to observe and study a single species of bacteria. A pure culture containing only one species of bacteria is ideally formed from one parent cell.7 This culture can be isolated by various techniques, as accomplished in this laboratory. A mixed culture containing two bacteria, E. coli and S. aureus were isolated by diluting the cultures using a streak plate method on solid media.2

   The isolation of bacteria is often accomplished using inoculating tools. Inoculation is the act of introducing a microorganism into a culture medium.8 Inoculating tools may need to be sterilized between every use or are pre-sterilized and must be disposed of properly after a single use.2 For the purpose of this laboratory experiment, the inoculating loop was not pre-sterilized. In order to avoid contamination, the inoculating loop must be sterilized between and after each transfer of bacteria as well as when the loop is contaminated by a surface.8 This is done by inserting the loop into a microincinerator for several seconds, and then letting it cool. The inoculating loop collects a loop full of culture from a broth culture which can then be transferred to a sterile medium.8 A glass spreader must be sterilized before and after use as well, however a pre-sterilized form was used in this laboratory. This is done by washing it with ethanol, and then igniting the residual ethanol.2 Other inoculating tools such as swabs, and pipettes are wrapped and pre-sterilized.2 These tools can only be used once and must be disposed of into the biohazard waste container after a single use. These tools must be unwrapped with caution, ensuring only the handle portion is touched, and not the portion collecting bacteria culture.2

   Several plating methods are used to isolate, propagate, and grow bacteria.9 These methods each incorporate aseptic technique, as discussed previously. Three major methods used are streak-plating; used to isolate single colonies, pour-plating; used for counting the number of colony-forming bacteria, and spread-plating; used to enumerate viable bacterial colonies.2 The streak-plate method is effective for the isolation of pure cultures from mixed populations.9 Although a single cell is not visible to the naked eye, a colony, presumably from one parent cell is visible and one can examine its characteristics.9 A nutrient agar plate is divided into four sections and an inoculating loop with bacteria is spread across each section.2 The concept being that each section of the surface will contain fewer bacterial cells deposited at widely spread points.2 Following incubation, separate colonies can form.2 Regarding the pour plate method, the bacteria are not limited to the surface of the agar, but rather are spread throughout as the agar solidifies around the bacteria.9 This process results in colonies distributed throughout the agar, making it possible to count the colonies as well as examine their morphology.9 Lastly, the spread plating method is commonly used in combination with a dilution series for enumeration purposes.9 A dilution series dilutes a concentrated population of bacteria by repeatedly transferring a known volume of bacteria- broth solution into a sterile test tube.2 The number of cells in the original sample can then be identified with the following equation:

(CFU/mL)=(number of colonies)/(Dilution factor × inoculated volume)

A sample from a test tube with a known dilution series can be inoculated onto an agar plate and spread using a glass spreader.2 Once incubated, colonies can be counted in order to identify the number of cells in the original sample. Each method is effective for various different purposes; however, all three plating methods maintain aseptic technique throughout.

Materials and Methods

Experiment 1.1-Ubiquity of Microorganisms

Part A: Airborne Microorganisms

   Equipment was set up as in BLG151 manual and the bench was wiped. A nutrient agar plate was exposed to air for the entirety of the lab. After the lab, the bottom of the plate was labelled with the date, names, and “1.1-Exposed to Air”. The closed petri dish was placed upside down.

Part B: The Swab/Swab Rinse Method

   Aseptically, the sterilized swab was unwrapped. A sample of the cheek was obtained. The swabbed sample was inoculated onto the nutrient agar petri dish. The petri dish was labelled with the date, names, and “1.1 Part B- Mouth Swab”. The closed petri dish was placed upside down.

Experiment 1.2- Aseptic versus Non-aseptic Techniques

Part A: Inoculation of tube with nonsterile inoculating loop

   A non-sterilized inoculating loop was touched against one’s fingers. The loop was inserted into a nutrient broth tube to the middle portion of the broth. The loop was removed, and the cap was replaced. The tube was labelled with names, the date, and “1.2 Part A Tube A”.

Part B: Inoculation of tube with sterile inoculating loop

   The inoculating loop was inserted into the microincinerator for ten seconds and removed to cool for ten seconds. With care to not contaminate the loop, the cap of the second nutrient broth was removed and the loop was inserted into the tube, to the middle portion of the broth. The loop was removed, and the cap was replaced. The tube was labelled with names, the date, and “1.2 Part B Tube B”. It was then set aside for incubation.

Experiment 1.3- Aseptic Inoculation of Broth Cultures

Part A: Inoculation using a sterile inoculating loop

   Four test tubes of nutrient broth were labelled. Tube 1 and 2 were labelled “control with loop”. Tube 3 and tube 4 were labelled with the bacteria Escherichia coli (E. coli) and bacteria Staphylococcus aureus (S. aureus), respectively. Upholding aseptic practice, a loop of broth was transferred from tube 1 to tube 2. The loop was sterilized and placed down. After re-sterilizing, a loop of E. coli broth was inoculated into tube 3 by repeating the method aforementioned. The loop was sterilized again, and a loop of S. aureus broth culture inoculated into tube 4 by repeating the method aforementioned.

Part B: Inoculation using a sterile pipette

   Four tubes of nutrient broth were labelled from 5-8. Tube 5 and 6 were labelled “control with pipette”. Tube 7 and 8 were labelled with E. coli and S. aureus, respectively. A sterilized 1.0mL pipette and pi-pump were used to transfer 1.0 mL of broth from tube 5 to tube 6. The pipette was discarded into the biohazard waste. 0.1 mL of E. coli broth culture was transferred to tube 7 with a new pipette, which was then discarded. This was repeated with S. Aureus and tube 8.

Experiment 1.4- Aseptic Inoculation of Solid Agar Media and the Streak Plate Technique

Part A: Inoculation of Nutrient Agar Slopes

   Two tubes of nutrient agar slopes were labelled with names, the date, and strains- E. coli and S. aureus. A loop of E. coli was aseptically obtained and transferred to sterile nutrient agar slope with a corresponding label. The loop was moved in a zig-zag pattern from the bottom to the top of the slope. This was repeated with the S. aureus broth.

Part B: Streak Plate Technique

   A nutrient agar plate was labelled with four sections, the date, name, and “1.4 Part B E. coli”. Using aseptic technique, a loop of E. coli was obtained from broth culture and used to inoculate the nutrient agar plate using the streak plate technique. This method was described on page 7 figure 1.4 of BLG 151 lab manual. This technique was repeated with S. aureus.

Experiment 1.5- Isolation of Pure Culture from a Mixed Population

   A nutrient agar plate was labelled with names, the date, and “1.5- mixed culture”. The streak plate method described by figure 1.4 was repeated with a mixed culture of bacteria.

Experiment 1.6- Streak Plate using a Solid Culture

   Using the plate from experiment 1.5, the bacteria were identified based on characteristics. Two nutrient agar plates were each labelled with one of the bacteria, and names. The streak method described in 1.4 was used to streak E. coli onto the plate labelled E. coli. The same was repeated with S. aureus from well isolated colonies.

Experiment 1.7- Preparation of a Dilution Series

   Five test tubes were labelled with their respective dilution series, as shown on page 10 of BLG 151 lab manual in figure 1.5. 1.0 mL of E. coli was pipetted into tube 1 and mixed. Using a new pipette, 1.0 mL of solution was transferred from tube 1 to tube 2, then gently mixed. This was repeated up to tube 5, creating a dilution series.

Experiment 1.8- Spread Plate Technique

   Using a sterile pipette, 0.1 mL of broth culture were transferred from tube 4 to a labelled nutrient agar plate. A sterilized glass spreader was used to spread the inoculum over the surface of agar. The spreader was placed down. This procedure was repeated with tube 5.

Experiment 1.9- Pour Plate Technique

   Broth from dilution tube 4 was pipetted (0.1 mL) into an empty petri dish. This was repeated with 0.1 mL from dilution tube 5. Quickly and carefully, half the molten agar content was poured into each petri dish and both petri dishes were swirled and set upside down. Observations were recorded for each culture and clean up procedure was followed as detailed on page 11 of the BLG 151 lab manual. All cultures were incubated upside down at 37ï‚°C.

Results

Referring to experiment 1.1, the nutrient agar plate in part A that was exposed to air for the duration of the laboratory experiment displayed three types of microbes on the plate. This is identified by the different colors of the colonies. There are two orange colonies, nine yellow colonies, and three white colonies. All of the colonies present are well isolated and round. The yellow and white colonies are flat. The orange colony is slightly raised. The agar media remained unchanged. The nutrient agar plate in part B is a mouth sample obtained with a sterilized swab. There are 20 white colonies. The colonies are flat, round, and well isolated. The agar media remained unchanged.

Referring to experiment 1.2-part A, tube A- inoculation with non-sterile loop did not display any bacterial growth and thus was not turbid. Part B, tube B- inoculation with sterile loop did not display any growth as well, and thus was not turbid. In experiment 1.3, test tube 1 and test tube 2, the control tubes with a sterile loop, did not display any growth and were not turbid. Test tube 3, containing E. coli, showed pellicle growth pattern in the broth media and it was turbid. Test tube 4, containing S. aureus was turbid and contained sediment. Test tube 5 and test tube 6, the control tubes with a sterile pipette, did not display any growth, and thus did not display any turbidity. Test tube 7, containing E. coli displayed flocculent growth patterns and turbidity. Test tube 8, containing S. aureus displayed sediment and turbidity.

Referring to experiment 1.4- part A: the agar slope containing S. aureus displayed effused and beaded growth patterns, with opaque, white bacteria growth. The agar slope containing E. coli displayed effused, translucent, shiny, and off-white in color bacterial growth. In part B, the growth of S. aureus bacteria on the agar plate was small, slightly raised, opaque, in isolated colonies and white in color. The growth of E. coli bacteria on the agar plate was large, round with slight spreading, fairly continuous in growth, shiny, translucent, and white-beige in color. There is a single colony of contamination on section 4 of the agar plate. This colony appears round, slightly raised, and yellow in color, as evident in figure 1.1.

Figure 1.1- Contamination of the 1.4- E. coli nutrient agar plate

In experiment 1.5 the mixed agar plate contains isolated colonies of E. coli and S. aureus. The E. coli appears large and white-grey in color with a continuous growth pattern. The S. aureus on the other hand appears small and round, and white in color. In experiment 1.6- the plate containing S. aureus lacked well isolated colonies. The bacteria present appear to be white, flat, and opaque. The plate containing E. coli appears to have continuous growth with translucent, white, and flat bacteria. Both plates did not display growth of the other bacteria.

Table 1.1- Comparing colony forming units/mL from two dilution series of E. coli

1.8- Spread Plate Technique 1.9- Pour Plate Technique

 Tube 4- (10-4) â–ˆ((CFU/mL)=(number of colonies)/(Dilution factor × inoculated volume)  )

(CFU/mL)=252/(10^(-4)  Ã— 0.1mL)

(CFU/mL)=25.2 × 106 â–ˆ((CFU/mL)=(number of colonies)/(Dilution factor × inoculated volume)  )

(CFU/mL)=157/(10^(-4)  Ã— 0.1mL)

(CFU/mL)= 15.7× 106

Tube 5- (10-5) â–ˆ((CFU/mL)=(number of colonies)/(Dilution factor × inoculated volume)  )

(CFU/mL)=39/(10^(-5)  Ã— 0.1mL)

(CFU/mL)=39.0 × 106 â–ˆ((CFU/mL)=(number of colonies)/(Dilution factor × inoculated volume)  )

(CFU/mL)=28/(10^(-5)  Ã— 0.1mL)

(CFU/mL)=28.0 × 106

Regarding experiment 1.8, both agar plates displayed isolated colonies of E. coli growth. The bacteria appear to be large, round, flat, and white in color. Tube 4 displays significantly more growth than tube 5. The pour plate technique in experiment 1.9 displays similar growth patterns. The E. coli bacteria appear to be round, white, translucent, with a slight spreading pattern. The pour plates also display lenticular colonies, embedded in the agar. Tube 4 displays significantly more colonies than tube 5.

Discussion

   Experiment 1.1-part A and part B results displayed the ubiquity of microorganisms. With reference to results question one, there were three colonies present on the agar plate that remained exposed to air for the duration of the experiment. Bacteria exist in a wide variety of environments, including the air in a laboratory setting. These bacteria may come from various sources including people’s skin, and coughing. The extensive growth on the agar plate supports the notion that contamination of sterile equipment is possible from simple exposure to the environment. The agar plate then provides the necessary nutrients for bacteria growth to flourish. Thus, it is essential to limit exposure of equipment by keeping media closed between inoculation and being aware of exposure once a sterile tool is unwrapped. Growth on this plate was generally expected. The agar plate containing a swab of the mouth contained only one type of colony, as described in results question one. The presence of bacteria in one’s mouth are expected, as bacteria can assist in digestion. The sample was retrieved from a person presenting with a cough. Accordingly, a presence of more strains of bacteria was expected than the results showed.

   Experiment 1.2-part A was the inoculation of a tube with a non-sterile inoculating loop. The loop was touched to fingers prior to inoculation. The tube displayed no growth, despite the lack of aseptic technique. This may have occurred because this person’s hands were washed prior to the laboratory, killing almost all bacteria present on their hands. Although these results were unexpected, they are understandable. The tube from 1.2- part B also displayed no growth. However, these results were expected as the inoculating loop was sterilized with a microincinerator prior to inoculation, killing all bacteria present on the loop.

   The control tubes in experiment 1.3 did not show any growth in the tubes. The control tubes, numbered 1 and 2 were inoculated by a sterile inoculating loop. As in experiment 1.2- part B, the inoculating loop was sterilized with heat application prior to inoculation. The lack of growth supports the concept of successful aseptic technique throughout the experiment. Test tube 3 (containing E. coli) and test tube 4 (containing S. aureus) did display growth of bacteria, as expected. This growth was made evident by turbidity in the tubes as described in results question 2. The control tubes numbered 5 and 6, inoculated by a sterile pipette did not display growth as expected. These results mean that no bacteria from the air were able to contaminate the pipettes between unwrapping and inoculation, supporting the success of aseptic technique in the laboratory. Tube 7 (containing E. coli) and tube 8 (containing S. aureus) displayed growth, as expressed by turbidity. Tube 3 and tube 7 appeared to have identical growth, as they contained the same bacteria. These results also occurred for tube 4 and tube 8.

  Experiment 1.4 tested one’s ability to aseptically streak nutrient agar slopes and the streak plate technique. The plate containing E. coli displayed the streak plate technique. There are several factors to note on this plate. Firstly, as displayed by figure 1.1, the agar plate displayed one colony of a different color than the rest of the colonies. This may be caused by contamination from either improper inoculation or from leaving the plate open for too long. The streak plate method is considered successful when isolated colonies are grown. As visible by the streaking pattern in figure 1.1, the inoculation loop did not streak the entire section, resulting in less separation of bacteria and thus fewer isolated colonies. In contrast, the plate containing S. aureus displayed a more effective streaking method, in which more colonies could be isolated. The inoculation of the agar slopes displayed differing growth between the bacteria, as described in results question 3.

   In experiment 1.5, the plate displayed a mixed population of E. coli and S. aureus. As described in results question 4, the bacteria differed in size, growth patterns, and color. This made it possible to isolate each colony for the inoculation of a pure culture on new agar plates from each bacterial colony for experiment 1.6. The isolation of pure colonies was completed successfully as each agar plate appeared to contain only one strain of bacteria.

When discussing the comparability of bacteria populations, it is important to keep in mind that bacteria are rapidly dividing microorganisms. Populations differing in even thousands of bacteria can be considered fairly comparable due to the enumerating nature of bacteria. Table 1.1 displays the calculations comparing the pour plate and spread plate techniques for two different dilution series. Regarding tube 4 with a dilution series of 10-4, the pour plate technique appeared to show 95 less colonies than the spread plate technique, resulting in a highly differing number for the number of colony forming units in the original sample. This differing number may simply be due to improper counting of colonies in the pour plate- technique agar. The colonies appear as thin lines in the agar and some may have been missed due to low visibility in the agar. Another possibility for the differing number is the fact that the pour plate method surrounds colonies in agar, limiting their access to oxygen.9 Aerobic bacteria cannot survive with limited access to oxygen, resulting in a smaller number of colonies in the pour-plate method plates.9 Lastly, the pipette may have also contained less bacteria for the pour plate method due to experimental error such as the bacteria settling to the bottom, rather than being in the middle of the broth during pipetting. The same pattern occurred for tube 5 with a dilution series (10-5). This raises suspicion that the same conditions occurred for both sets of agar plates. As expected, the more dilute test tubes resulted in a smaller number of colonies on the agar plates. Referring to the spread plate method, tube 4 contained 213 more colonies than tube 5. This result is expected as the bacteria are meant to be more diluted in tube 5 than tube 4 for the purpose of isolation of bacteria in a dilution series.

   Overall, each plating technique displays unique advantages and disadvantages. The spread plate technique limits the growth of anaerobic microorganisms but allows aerobic cultures to flourish.9 The streak plate method is effective in isolating distinct colonies and the observation of colonies, but it cannot be used for quantitative studies regarding the calculation of colony forming units in an original sample.9 As previously discussed, the pour plate method limits the growth of aerobic bacteria due to the lack of oxygen in agar.9 This method is effective for selectively growing anaerobic microorganisms.2

Conclusion

   The growth of bacteria is abundant and versatile.1 This was depicted by the exposed agar plate which grew a variety of bacterial colonies. For this reason, aseptic techniques must be applied and well understood in order to prevent contamination.3 Aseptic technique involves the practice of washing hands, the use of sterile tools, and disinfecting laboratory benches.2 The goal of aseptic technique is to maintain a contamination-free laboratory environment as depicted by the control group test tubes. The disposal of tools in the biohazard waste containers including pipettes also exemplifies aseptic technique.3 Still, the presence of bacteria may be a desired result in a science laboratory, as bacteria can serve several useful applications.5 As bacteria commonly occur in mixed populations with numerous strains present, several basic microbiological techniques are applied to isolate one strain into a pure culture through inoculation.7 Lastly, these isolation techniques differ between the streak plate technique, pour-plate technique, and spread-plate technique.9 Each method involves various media to allow bacterial growth.