Lab report Selective Differential mediums PDF

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Running head: Differential and Selective Medium

Analyzing Differential and selective medium on EMB and MSA

Jodyann munroe

Microbiology

7/31/2020

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Abstract This experiment report is made on differential and selective medium on Eosin Methylene Blue Agar and Mannitol Salt Agar and Tryptic Soy Agar used for the control. The introduction part gives an explanation on how bacterial takes place on various growth medium as well as how various bacterial strains and microorganisms respond to growth in mediums with varying components. It explains on the three types of mediums that’s the complex, selective and differential mediums. It further explains on the two types of sample organisms that the Aureus and E. Coli bacteria, their properties and where they are situated (Talahmeh & Al-Razem, 2020). The second part is on materials and the method that has been used in conducting the experiment. The results observation and results have been indicated on the third section and finally the forth section gives a vivid description and a deeper explanation on these responses and reasons behind the results that have been observed in the four medium plates. Every observation and result in every plate has a significant attachment with the properties of the medium of growth as well as the properties of the two microbes used in the experiment. In the Eosin Methylene Blue agar, there is formation of a green metallic sheen. Thad nature is due to the dye properties of the stains. The medium inhibits growth of gram positive bacteria and encourages the growth of gram positive bacteria. In this case, E. Coli is gram negative hence its growth is favored. There is also lactose fermentation that took place that resulted in te nature of the medium. In the Mannitol Salt Agar plate, there is formation of two regions that a yellow and a pink region separated. The yellow region is due to the fermentation of the S. aureus to produce lactic acid (Ashraf & Shah, 2011). The acid being acidic in nature turens the red medium indicator into yellow color. The pink region is due to the effect of other microbes. The other medium, that the Tryptic Soy Agar favored the growth of both E. coli as well as Aureus. This is because it has got all the necessary nutrients and constituents that favor the growth of a broad variety of bacteria and other nutrients, it is non-selective and hence was conducive for their growth. Introduction The growth of all microorganisms is dependent on favorable conditions of growth with the required nutrients to sustain its growth and survival. Bacterial growth need a medium of growth to support their growth. This is known as a culture medium or a growth culture which is a

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liquid, solid or a semi-solid constituent that aids the growth of cells or a population of microbes through the process of cell division and proliferation. Mediums vary as well, there are some that favor the growth of a particular microorganisms and others inhibit the growth of some. This is dependent on the nutrients that in present in a particular medium as well as the need of the microorganism. There exist selective, differential and complex medium (Ashraf & Shah, 2011). Selective medium selects for the growth of an organism of interest as it hinders or kills the growth of the undesirable microbe. Differential medium utilizes the biochemical proprieties that a target microbe possesses. Complex medium normally contains pure biochemical such as milk, blood, beef or yeast extract, the specific chemical composition is not well determined. Differential media is use in the differentiation of very closely related organisms, strains and species according to their biochemical characteristics and properties. Selective medium on the other hand allows only the growth of microbe that has been selected for example, in case a microbe is resistant to a particular antibiotic, such as tetracycline or ampicillin, then the addition of this antibiotic to their growth medium hinders growth of other cells which lack the resistance to enable its growth. There are many types of medium but in this report, EMB. MSA and TSA are going to be addressed. Mannitol Salt Agar(MSA) is a differential and selective growth medium commonly used in microbiology. It enhances growth of a particular group of bacteria whereas hindering the growth of others. MSA is used in the determination on whether the bacteria are halophilic or not and if whether the bacteria have the ability to ferment mannitol. In case the bacteria are able to growth, it means it’s a halophilic (salt loving) bacteria as a result of its ability to withstand growth in a salt concentrated environment. Eosin Methylene Blue(EMB) medium is a selective Gram-negative bacteria stain. The dyes in this medium are toxic to bacteria that is gram- positive. This medium serves both as a deferential and a selective medium of coliforms (Shen & Zhang,2017). It’s made up of an assortment if Methylene blue and Eosin in a 1:6 ratios. Eosin and Methylene blue are indicators that are used in the differentiation between lactose-fermenting bacteria and microbes that do not ferment lactose. Triplicate Soy Agar(TSA) is a medium used I culturing bacteria (Vessel, et.al., 2019). It’s a non-selective, general purpose media that provides sufficient nutrients to enable the growth of a broad a wide variety and strains of microorganisms to grown. Microorganisms that need vitamins or other factors to facilitate growth are able to grow on this media. The type, metabolic properties and constituents of

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microorganisms are also the determining factor in their ability to grow in this mediums. In this experiment, Staphylococcus aureus and Escherichia coli are inoculated into this medium. Staphylococcus aureus is a gram positive bacteria. It is considered the most dangerous among the commonly known varieties of staphylococcal bacteria. It I spherical shaped(coca) and a member of the Formicates bacteria and is commonly known to cause infections of the skin as well as causing heart valve infection, pneumonia and bone infections. It’s found in the nose, the skin, groin, armpit and other areas. E. coli is a gram-negative, rod-shaped, facultative anaerobic coliform bacterium that is mostly found in the intestines and in the gut of some animals. Majority of E. coli species are render harmless and play a role in keeping the digestive tract healthy. However, there are some strains that harmful that causes diarrhea id one drinks contaminated water or stale food. The aim of this experiment is to illustrate the uses of selective and differential media for the isolation as well as characterization of organisms of interest. It also shows how the two microbes E. coli and Aureus respond on being inoculated into the Eosin Methylene Blue agar, Mannitol Salt Agar and Tryptic Soy Agar. Its hypothesized that Staphylococcus aureus would grow on the Eosin Methylene Blue Agar since it its gram negative and the medium favors growth of the gram negative bacteria. Also E. coli would grow on Mannitol Salt Agar and its expected there would be a change in the medium color due to the fermentation ability of the E. coli on lactose present (Wallace & Jones, 1996).

Materials and Method Prepare medium of Eosin Methylene Blue, Mannitol Salt Agar and Tryptic Soy Agar medium in a conical flask under sterile aseptic conditions. Stir well to ensure a complete dissolution of the powder. For each medium, pour into its specific culture medium one for each and for Tryptic Soy Agar pour into two separate mediums. Cover them well and place them into an incubator at 37 degrees Celsius for 48 hours. After 48hours, introduce the sample microbes into the mediums while working in a sterile environment and sterile bench. Our provided with samples of Staphylococcus aureus and Escherichia Coli. Sterile inoculation loop using a flame. Using the wire loop, scoop a colony of Staphylococcus aureus and streak it on the surface of Mannitol Salt Agar plate and one of Tryptic Soy Agar plates. Cool the loop then do the 2 nd streaking and make the 3rd streaking. Single colonies will be visible on the 2nd or the 3rd streak

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depending on original sample density. The sterilize again

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and inoculate E. coli in Eosin

Methylene Blue Agar plate and the other plate of Tryptic Soy Agar plate. Once done, place the inoculated plates in an incubator at a temperature of 37 degrees Celsius for 48 hours. Then make observation and record the results.

Observation and Results In the Eosin Methylene Blue Agar plate, E. coli appears large. Colonies that are blue black with a green metallic sheen (Shen & Zhang,2017). In the Mannitol Salt Agar Plate, there is formation of two zones, one is yellow and the other pink. S. aureus gives yellow colonies with yellow zone surrounding it. In the two Tryptic Soy Agar plate, there was growth of colonies of E. coli and Aureus respectively.

Discussion Mannitol Salt Agar is earlier indicated is a selective medium which plays a role in the pathogenic staphylococci isolation. Mannitol is among the constituents of the medium, it’s a phenol red indicator and is 7.5% made up of Nalco (Wallace & Jones, 1996). Most bacteria growth is inhibited in this this medium due to its high concentration of salt other than staphylococci which are halophilic bacteria. On MANNITOL Salt Agar, Aureus which is pathogenic produces small yellow colonies within a yellow region. This is because Staphylococcus aureus ferments mannitol, an acid byproduct is produced which subsequently changes the red indicator to yellow color. The other varieties of bacteria are inhibited. Eosin Methylene Blue Agar being a selective medium favors the growth of gram negative bacteria and hinders the growth of gram positive bacteria. The Eosin Methylene Blue contains stain that renders gram positive bacteria. The prominent metallic green sheen observed is as a result of the dyes metachromatic properties and also due to the robust acid end product test of fermentation. Escherichia Coli is a lactose fermenting bacteria hence its fermentation result in the formation the observed growth and color. The Eosin and Methylene blue are dyes that have

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aided in the identification and the observation of the properties and the response Staphylococcus aureus.

On the Tryptic Soy Agar plate, there is a complete growth of the two bacteria colonies. The medium ere used is for general purpose and hence has all the vitamins and nutrients that favor the growth of many microorganisms. The streaks are very distinct and have a continuous growth. Here, there is no inhibition of whichever kind hence both gram positive and gram negative bacterial growth is encouraged (Welch et.al.,1987). The metabolic properties of the microbes do not negatively affect the growth of the medium since there is no stay nor dies that can be toxic or hinder their growth. In this experiment, it is clear that there are some properties that a microorganism possess that may be suitable or unsuitable to enable its growth in various mediums. Additionally, selective and differential medium is ideal for the identification of some particular group of organisms due to their selective nature. In conducting test and during research, selective and differential mediums are important to enable isolation, characterization and identification of properties and nature of some organisms (Shen & Zhang,2017). The general purpose medium that’s Tryptic Soy Agar does not enable the identification of a specific microorganism but it is important when there is the need of growing many varieties of organisms for study or for other purposes. Since there are millions and discovered microorganism, various distinguishing mediums ought to be formulated so as to enable each and every microorganism to be identified and understood on the basis of its properties, characteristics, physiological as well as metabolic pathways and properties. This advancement enables advanced discoveries and improved control and usage of microorganisms.

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References Ashraf, R., & Shah, N. P. (2011). Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt—A review. International journal of food microbiology, 149(3), 194-208. Talahmeh, N., Abu‐ Rumeileh, S., & Al‐ Razem, F. (2020). Development of a selective and differential media for the isolation and enumeration of Bacillus cereus from food samples. Journal of Applied Shen, C., & Zhang, Y. (2017). Isolation of Foodborne Pathogens on Selective, Differential, and Enriched Medium by Streak Plating. In Food Microbiology Laboratory for the Food Science Student (pp. 19-23). Springer, Cham.Microbiology, 128(5), 1440-1447. Wallace, J. S., & Jones, K. (1996). The use of selective and differential agars in the isolation of Escherichia coli O157 from dairy herds. Journal of applied bacteriology, 81(6), 663-668. Welch, D. F., Muszynski, M. J., Pai, C. H., Marcon, M. J., Hribar, M. M., Gilligan, P. H., ... & Chartrand, S. A. (1987). Selective and differential medium for recovery of Pseudomonas cepacia from the respiratory tracts of patients with cystic fibrosis. Journal of Clinical Microbiology, 25(9), 1730-1734. Veselá, K., Kumherová, M., Klojdová, I., Solichová, K., Horáčková, Š., & Plocková, M. (2019). Selective culture medium for the enumeration of Lactobacillus plantarum in the presence of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. LWT, 114, 108365....