Isolation and Identification of Bacillus megaterium,
Escherichia coli, and Providencia rettgeri
Introduction
Bacteria or usually known as a unicellular microorganism that lacks a membrane-bound nucleus [1]. Bacterial cells are very small and are unable to be seen with the naked eye but with the use of a microscope [1]. Bacteria exist in various cellular morphology. Bacteria are said to exist in different size, shape, arrangement and cell organization such as the presence of the bacterial cell wall and the flagella that may give rise to different characteristics. Although the bacterial are relatively small and simple it is can usually appear as cocci, rod, comma or even sphere shaped [1]. Arrangement such as a singular bacterial cell, pairs, cluster or even chains can be observed under the use of a microscope.
Bacteria can be found in many different places of the environment and the human body [1]. Bacteria contributes to a balanced ecosystem and a healthy development of the human immune system. However, some bacteria could cause serious damage to place it inhibits – such as the human body. Therefore, it is important to identify and isolate a bacterial cell to determine its pathogenicity and the effects of it on the human body.
To identify a bacterium, it must first be isolated from the mixed culture that contains more than one type of unknown microorganism growing in the medium. To do so, the streak plate method is used. Streak plate is used to separate the different colonies of microorganisms to obtain a pure culture by diluting the initial sample [1]. In this experiment, sample is transferred using an inoculating loop from a glass bottle and onto the agar plate. Before that, the inoculating loop is first passed through a burning flame to sterilize and to remove any residue that may contaminate the culture which is known as an aseptic technique [1]. The inoculating loop is then used to distribute the sample around the surface of the nutrient agar. The inoculating loop is again passed through the flame after every streak that was made. The same inoculating loop is then used to distribute the mixed culture sample onto the MacConkey agar. This is conducted to prevent the contents from the MacConkey to be transferred to the nutrient agar.
A pure culture or an isolated colony of bacteria can be cultivated on different type of mediums. Different medium can be used to cultivate different types of organism depending on the conditions exposed during incubation. A growth medium can be a selective, differential or both. Selective media only allows growth of certain microorganisms and inhibits the others. A type of media that is both selective and differential is the MacConkey agar containing bile salts and crystal violet is differential towards the growth of Gram-negative bacteria and Gram-positive cocci and rods. Lactose producing bacteria (lac+) allows it to be differentiated due to its characteristics and the neutral red – a pH indicator. This allows easier identification as the lactose is used as a carbon and a source of energy [1]. Colonies that releases acid can be seen to be red in colour while the other microorganism are colourless. The MacConkey agar can be made to be more selective by incorporating different type of bile salts into the agar. Example of differential media is the blood agar which is differential towards partial or complete hemolysis that is causes by the protein produced from the bacteria [1]. However, in this experiment, the MacConkey agar and the nutrient agar is used to cultivate the bacterial cells. Nutrient agar contains sufficient nutrients for the growth of different type of microorganisms such as bacteria and fungi. Agar is then left to incubate at its respective temperatures.
After the process of cultivation of bacteria, the respective agar plates can then be observed for the colony morphologies such as the appearance, arrangement and the colour of the colonies as well as the morphologies of the bacterial cell. Both type of agar is also observed to see if the media can sustain the growth of the bacteria. Apart from that, gram staining is used to stain the bacterial cell for the process of identification. This allows the shape, arrangement and the structure of the cell wall to be observed. The gram staining is a differential staining process based on the characteristics of the bacteria [1]. The stain differentiates the Gram-positive and the Gram-negative microorganisms. Gram staining is done by firstly preparing a heat fixed smear with a small sample of the isolated pure culture. This is to make sure the stain can be retained well within the cell and to adhere the sample on to the glass slide. Then, the process is followed by the adding of Crystal violet stain for about 30 seconds then rinsed with water. This process is repeated with Gram’s iodine, alcohol and diluted carbol fuchsin. Gram’s iodine is important to help retain the dye for stronger staining [1]. The Gram-positive bacteria are stained purple while the Gram-negative bacteria are stained pink [1]. This is due to the peptidoglycan layer of the bacterial cell. Gram-positive bacteria with a thick peptidoglycan layer can retain the purple crystal violet dye within the peptidoglycan layer. Gram-negative bacteria however have a thin peptidoglycan layer and is unable to retain the crystal violet stain therefore it is stained pink from the diluted carbol fuchsin.
Aim(s)
The aim of this experiment is to isolate the different types of microorganisms using nutrient agar and the MacConkey agar. Both the medium is used for different purposes. After incubation, the different types of microorganisms are then able to be identified by their appearance, colour, colony type and the bacterial morphology.
Materials and Methods
As per the MIC2011 Introduction to Microbiology and Microbial Biotechnology Practical Class Notes 2018, page 61. The methods on preparation of smear and Gram staining can be referred in the Microbiology Techniques Manual 2018 on page 27-28. Methods on streak plate can be referred in the Microbiology Technique Manual on page 40.
Results
This experiment was conducted within the duration of 2 weeks. Gram staining was performed using a mixed culture. This was conducted to observe the bacterial morphology. Then, sample of mixed culture was distributed on the nutrient then the MacConkey agar using the same inoculum loop according to the streak-plate technique. This is to obtain a pure culture as a product after incubation. Plates are then stored at 4°C till the day before the second session. The samples are then taken out to be incubated at 37°C for 24 hours. The conditions of the cells on both the agar are observed. Small sample of the cells are taken to carry out Gram-staining. Observation from both the initial Gram stain and final Gram stain is compared. Results are tabulated.
Table 1: Table of the colony morphology and the cellular morphology of the different types of organism on the nutrient agar and MacConkey agar which was stored at 4°C up till the day before the next lab session, which then placed to incubate at 37°C for not more than 24 hours.
Type of Organism
Type of Medium
Incubation conditions
Appearance
Cellular Morphology
Nutrient Agar
MacConkey Agar
Shape
Arrangement
Gram Stain
A
Growth with white colonies
No growth
Stored at 4°C then incubated at 37°C for 24 hours
Round
and shiny
Rod
Chain
Positive
B
Growth with white colonies
Growth with red colonies
Stored at 4°C then incubated at 37°C for 24 hours
Round and smooth
Rod
Random
Negative
C
Growth with white colonies
Growth with beige colonies
Stored at 4°C then incubated at 37°C for 24 hours
Opaque, shiny
and convex
Rod
Random
Negative
The results showed three types of pure bacterial culture was isolated from the mixed broth culture. All three bacterial cell types grew on the nutrient agar and all three of the colonies were white in colour. However, only microorganism B and C grew on the MacConkey agar. Microorganism B grew red colonies while microorganism C grew beige colonies on the MacConkey agar. All three bacteria cells showed differences in term of the morphology of the colonies. Colonies of microorganism A had a round, shiny appearance to it. Colonies of microorganism B were round and smooth. Colonies of microorganism C were opaque, shiny and appeared to be convex. All three microorganism were rod-shaped. Microorganism A had a chain arrangement while microorganism B and c had random arrangements. Only microorganism A was positive gram-stained while the other two – microorganism B and C showed negative-gram staining.
Discussion
From the results, it can be identified that microorganism A is the Bacillus megaterium. From the table, it can be said that the Bacillus megaterium grew as white, round and shiny colonies that is only able to grow in the nutrient agar and not the MacConkey agar. MacConkey agar remained transparent. This is due the different properties of both the agar. It can grow large amounts of pure culture on the nutrient agar as it contains plentiful nutrients to support growth of various types of microorganisms. Agar is widely used in the isolation of microorganism as it remains its solid state and will only melt at a very high temperature [1].. This is due to the MacConkey agar being both selective and differential to the microorganisms that could be cultivated on it [1]. MacConkey agar only supports the growth of enteric bacteria. Enteric bacteria are gram negative rod-shaped bacteria usually found in the intestines of a living organism or is commonly known as the gut flora [1]. On the other hand, non-enteric bacteria are bacteria which are not found within the intestinal tract. This shows that the Bacillus megaterium is a non-enteric bacterium and does not live within intestines of humans or animals. Bacillus megaterium are usually found inhibiting areas with soil – living of the nutrients from decaying organic material from the soil [3]. Upon gram staining, the slide is observed with the use of the microscope. From the slide, it can be observed that the Bacillus megaterium is a rod-shaped bacterium that usually exists in chains. The bacteria showed positive gram-staining. This shows that the bacteria have a thick peptidoglycan layer as it can retain the crystal violet dye well within its cell wall [1].
Apart from that, MacConkey agar can increase its selectivity by the addition of either bile salt no. 2 or bile salt no.3. Addition of bile salt no.2 when added, it supports the growth of Enterococci bacteria [2]. However, if bile salt no.3 is chosen, the growth of all types of gram positive bacteria is inhibited.
According to the results that was tabulated above, it can be said that microorganism B is Escherichia coli. The Escherichia coli grew as white colonies on the nutrient agar and red colonies on the MacConkey agar. The MacConkey agar is important to differentiate lactose fermenting (lac+) from non-lactose fermenting bacteria (lac-). The agar is said to be selective as it consists of lactose, bile salts and crystal violet that inhibits the growth of gram-positive bacteria [1]. Neutral red acts as an indicator for when the pH of the environment changes as it turns the agar pink when the pH decreases. This shows that Escherichia coli can ferment lactose (lac+) and is an enteric bacterium. The Escherichia coli bacteria is usually found in the intestine of both human and animals. It is also the cause towards severe food poisoning if any uncooked food is consumed [4]. The lactose from the agar is fermented by the Escherichia coli to be used as a source of carbon and energy [1]. Although the Escherichia coli carries out aerobic respiration, it can switch to anaerobic respiration or fermentation in the absence of oxygen. The lacZ gene is the structural gene for β-galactosidase which activates the lac operon when amount of glucose within the cell is low in the presence of lactose [5]. The appearance of the red colonies and the pink agar is from the acidic end-products from the lactose fermentation such as carbon dioxide. This also causes the bile salts to precipitate around the growth of the Escherichia coli. From the agar, it can be observed that the Escherichia coli appears to be round and smooth. Upon staining and observing, it can be said that the Escherichia coli is rod-shaped and has a random arrangement. It is also gram-negative bacteria. This shows that the Escherichia coli has a thin peptidoglycan layer thus it is unable to retain the crystal violet dye and is decolorized when alcohol is applied to it. The pink stain to the peptidoglycan of the cell comes from the counterstain of the carbol fuschin.
Microorganism C is identified to be Providencia rettgeri. It can grow in both nutrient and MacConkey agar. This shows that the Providencia rettgeri is an enteric bacterium. It is usually found in the urinary tract and the intestinal tract of human beings and animals. However, the Providencia rettgeri is unable to ferment lactose giving it the lac- properties. Although it can grow on the MacConkey agar, it does not change the colour of the pH indicator and no lactose is being fermented to release any acidic end-products such as carbon dioxide. There is no lacZ gene that codes for β-galactosidase to activate the lac operon when there is lack of glucose in the cell. Thus, fermentation does not occur. This gives the beige colonies on the MacConkey agar. From the results, it shows the Providencia rettgeri has opaque, shiny and convex colonies. It is a rod-shaped bacterium and has a random arrangement. When stained, the cells appeared to be pink indicating it is a gram-negative bacterium. This shows that the Providencia rettgeri has a thin peptidoglycan layer and is unable to retain the primary stain – crystal violet but retains the carbol fuschin.
In this experiment, it is important to first inoculate the mixed culture onto the nutrient agar then the MacConkey agar. This is done to prevent the bile salts from the MacConkey agar to be transferred to the nutrient agar. Transfer of bile salts may inhibit the growth of some microorganisms. This would give no difference to both the agar after incubation as the same type of microorganisms are isolated. Both agar plates would show same results. It would be hard to identify the initial types of microorganism that existed in the mixed culture.
It is also important to note that the MacConkey agar should not be incubated for more than 24 hours. This is to avoid the over usage of lactose for fermentation. For example, if the agar is incubated for too long, the Escherichia coli may have finished using up the lactose for fermentation. This may cause in fading of the pink colour of the pH indicator. This would yield beige colonies of bacteria instead of red. This way, it would be harder to identify and differentiate the bacteria.
Conclusion
Three types of bacteria were isolated using the streak-plate technique which is the Bacillus megaterium, Escherichia coli and the Providencia rettgeri. The Bacillus megaterium was identified being the only Gram-positive bacteria due to its structure. The Escherichia coli and Providencia rettgeri was identified based on the colony growth on the MacConkey agar the showed difference in colours.