Report 2 - Grade: B+ PDF

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Laboratory report of exercises listed in the document. Taken from the text Microbiology: theory and laboratory approach....

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Aseng 1

Report 2: Selective and Differential Media Exercises: 4-1, 4-3, 4-4, 4-5, 4-6, 5-2, 5-3, 5-20 Microbiology Lab 120:335:001 Jessica Aseng 29 October, 2019 TA Alexandra Adams

Objectives:

Aseng 2 Exercise 4-1. The objective of this exercise is to inoculate one selective phenylethyl alcohol agar (PEA) plate and nutrient agar (NA) plate with three organisms. Students will observe how well the organisms grow on the PEA plate compared to the NA plate and will determine the presumptive identification of the organisms. Exercise 4-3. Bile esculin agar (BEA) is a selective as well as differential medium that was used to isolate and determine the identification of several organisms to determine whether the plates can be recorded as positive or negative. This will establish if the organisms is a member of Group D Streptococcus or Enterococcus. Exercise 4-4. Mannitol Salt Agar plates are used to isolate and differentiate Staphylococcus aureus from other Staphylococcus organisms. It uses the ability of the organisms to grow on the plates as well as color changes to establish if the organism is part of the Staphylococcus group. Exercise 4-5. In this exercise, MacConkey agar plates are utilized to isolate and differentiate organisms of the Enterobacteriaceae family by each organism’s ability to ferment lactose. This is determined by the targeted organism’s growth and the color of the growth compared to a control group. Exercise 4-6. Eosin Methylene Blue agar, or Levine, is a selective and differential setting that inhibited the growth of most Gram-positive organisms and reacted with lactose fermenters. This process is used for the isolation of fecal coliforms. Exercise 5-2. The Oxidation-Fermentation test was used to classify bacteria by their ability to oxidize or ferment certain kinds of sugars. Exercise 5-3. The Phenol Red broth (PR), like Exercise 4-5, is used to differentiate Enterobacteriaceae, but it separates them from other Gram-negative rods. Students also use this exercise to distinguish between Gram-positive fermenters.

Aseng 3 Exercise 5-20. SIM media (Sulfur reduction, Indole reduction, and Motility) are used to analyze bacteria that can produce indole as well as differentiating sulfur-reducing organisms of Enterobacteriaceae. Motility is also determined in the exercise.

Introduction: Exercise 4-1 Phenylethyl alcohol agar (PEA) is a selective medium that will grow Gram-positive organisms. Selective media supports the growth of some organisms, most Gram-positive bacteria, while inhibiting the growth of others, Gram-negative organisms (Leavall, Sandie, et al, 2007). While some media can be differential, PEA is not a differential environment because it cannot make distinctions between the organisms that are able to grow on its plates. In order to inhibit Gram-negative bacteria, PEA alters the permeability of the bacteria’s membrane which lets in molecules that would have otherwise been blocked. This also allows cellular potassium to leave the cell, which disrupts the DNA synthesis of Gram-negative bacteria, thus inhibiting their growth on these cultures (Lal & Naowarat, 2011). Students spot-inoculated one PEA plate and another nutrient agar (NA) plate with three organisms: Escheria coli (EC), Enterococcus raffinosis (Er), and Staphylococcus saprophyticus (Ss). The NA plate was used as a control group to compare the growth quality one the PEA plates. After incubating the plates, students are to observe the growths on the plates. If there was poor growth or no growth on the PEA plate compared to the NA plate, the organism is most likely Gram-negative and was inhibited by the phenylethyl alcohol. If there is good growth viewed on the PEA plate than the organism was not inhibited and is Gram-positive (Leboffe & Pierce, 2015).

Aseng 4 Exercise 4-3 Bile esculin agar (BEA) is a selective and differential media made of beef extract, digest of gelatin, esculin, oxgall, and ferric citrate. Esculin is derived from the horse chestnut tree and is a glycoside made of glucose and esculetin. The oxgall component is the selective agent that is part of the agar separating Group D streptococci from other non-Group D streptococci. Ferric citrate is also added to show a positive test. Overall, BEA is used to presumptively identify enterococci and group D streptococci based on the ability of an organism to hydrolyze esculin (Bile Esculin Agar, 2019). Esculin hydrolysis produces D-glucose and esculetin. There are many bacteria that have esculinase, but bacteria with the ability to hydrolyze esculin with bile are much smaller in number, which makes this process an effective selective and differential medium (Leboffe & Pierce, 2015). In this exercise, BEA prepared as a plate to isolate streptococcus or enterococcus from a mixed culture. Once the plate is prepared, esculin is hydrolyzed in BEA which allows esculetin to react with ferric ions generating a dark colored growth. Observing a plate that is dark in color produces a bile esculin positive result. If the organism does not darken the medium, then it is negative. Students used three organisms for observation: Lactococcus lactis (Ll), Enterococcus raffinosis (Er), and Providencia alcalifaciens (Pal). If the medium is darkened after 48 hours, then the organism is identified positively as a member of the Group D Streptococcus or Enterococcus. No darkening of the medium results as a negative which deems the organism as not a member of the Group D Streptococcus or Enterococcus. Exercise 4-4 Mannitol Salt Agar (MSA) is another type of selective and differential medium. It is

Aseng 5 made up of carbohydrate mannitol, 7.5% sodium chloride, and the pH indicator phenol red which turns different colors at different levels of pH. Phenol red is yellow below pH 6.8, red between pH 7.4 to 8.4, and pink above pH 8.4 (Leboffe & Pierce, 2015). The mannitol component allows for fermentation and makes the medium differential. The high concentration of salt selects for organisms that can tolerate high saline levels, and kills off bacteria that will dehydrate in this environment (Leavall, Sandie, et al, 2007). Staphylococci will survive in the salty medium because of their tolerance to salty environments. Phenol red expressed whether fermentation with an acid end-product has taken place by changing color as the pH shifts. Many staphylococci organisms are able to grow on MSA, but are not able to ferment mannitol, so their medium color remains unchanged at pink or red. Staphylococcus aureus is able to ferment mannitol, lowering the pH of the medium and forming yellow colonies with a yellow halo. Students spot inoculated one MSA plate and one nutrient agar (NA) with four organisms: Staphylococcus saprophyticus (Ss), Staphylococcus epidermidis (Se), Escherichia coli (Ec), and Enterococcus raffinosus (Er). Observations of the plates after incubation will be recorded in tables; poor growth or no growth means that the organism was inhibited and is not Staphylococcus, organisms with good growth were not inhibited by NaCl and are Staphylococcus, yellow growth or a halo yields organisms that produce acid from mannitol fermentation, and red growth with no halo means that the organism does not ferment mannitol. Exercise 4-5 MacConkey Agar (MAC) is a selective and differential medium that is used to isolate and differentiate organisms based on their ability to ferment lactose (Leavall, Sandie, et al, 2007). This medium also contains bile salts, neutral red, and crystal violet. The bile salts and crystal violet inhibit growth of Gram-positive bacteria. Neutral red dye is a pH indicator that is colorless

Aseng 6 above a pH of 6.8 and red at a pH below 6.8. Bacteria that are able to ferment lactose lower the pH and their growth turn a pink or red color. Bacteria that cannot ferment lactose remain their normal color or retain the color of the agar. MAC is used to isolate and differentiate organisms in the Enterobacteriaceae family. Students will be spot inoculating one MAC plate and one NA plate with five organisms: Enterococcus raffinosus (Er), Escherichia coli (Ec), Providencia alcalifaciens (Pal), Klebsiella aerogenes (Ka), and Citrobacter freundii (Cf). After incubation, the growths and colors of the colonies will be compared to the NA plate and the results recorded: poor growth or no growth yields organisms that are inhibited by crystal violet or bile and is gram positive, good growth of bacteria means the organisms are not inhibited and are gram negative, pink to red growth means organisms produce acid from lactose fermentation and are probable coliform, and, finally, growth that is colorless yields organisms that cannot ferment lactose and are non coliform. Coliform is a term used to describe bacteria that are present in nature and are found in all human and animal waste.

Exercise 4-6 Eosin methylene blue (EMB) agar is a selective and differential medium containing digest of gelatin, lactose, and the dyes eosin Y and methylene blue (Leavall, Sandie, et al, 2007). Gelatin provides organic carbon and nitrogen. The dyes have two functions in this agar: inhibiting the growth of most Gram-positive organisms and reacting with lactose fermenters who products turn dark purple or black. The dark bacteria is a typical sign of E. coli and is also accompanied by a green metallic sheen. Other, less-aggressive fermenters, produce growths that can be pink or dark purple while lactose non-fermenters usually retain their normal color or keep

Aseng 7 the color of the medium. Students will be spot inoculating one EMB plate and one NA plate with five organisms: Enterococcus raffinosus (Er), Escherichia coli (Ec), Providencia alcalifaciens (Pal), Klebsiella aerogenes (Ka), and Citrobacter freundii (Cf). After incubation, students should see a range of characteristics that will determine the identification of the organisms: poor or no growth means the organism was inhibited by eosin and methylene blue and is Gram-positive, good growth yields organisms that were not inhibited and are Gram-negative, pink growth that is mucoid yields bacteria that ferments lactose with little acid production and is a possible coliform, growth that is dark also ferments lactose and is a more likely coliform, and, finally, growth that is colorless does not ferment lactose or sucrose and is non coliform. Exercise 5-2 The Oxidation-Fermentation (O-F) is a differential test that discriminates bacteria based on fermentative or oxidative metabolism of carbohydrates. Organisms that are oxidative in nature oxidize carbohydrates to CO2, H2O, and energy. They use glycolysis, oxidation of pyruvate, the citric acid cycle, and the electron transport chain. Similarly, fermentative bacteria convert the carbohydrate to pyruvate, except they do not use oxygen either because the cannot or it is not available; they have pyruvate act as the final electron acceptor and reduce it to an acid, gas, or alcohol fermentation end products (Leavall, Sandie, et al, 2007). The O-F medium also has a high sugar-to-peptone ratio to lessen the possibility that alkaline products from peptone utilization will neutralize weak acids produced by oxidation of the carbohydrate. Bromothymol is used as the indicator; it is yellow at pH 6.0, green at pH 7.1, and blue at pH 7.6. Low agar concentration makes the medium semisolid which allows students to determine the motility of the bacteria.

Aseng 8 The broths are prepared with glucose, lactose, dextrose, maltose, mannitol, or xylose, Two tubes are stab inoculated with the test organisms; then one tube is sealed with a layer of sterile oil to promote anaerobic growth and fermentation. This oil does not let oxygen diffuse into the medium, so oxidation cannot occur. The other tube is left unsealed so oxygen can seep into the medium. Bacteria that are able to ferment or ferment and oxidize the carbohydrate will turn the sealed and unsealed media yellow throughout. Organisms that are able to oxidize only will turn the unsealed broth yellow and let the sealed medium turn green or blue. Slow or weak fermentors will turn both tubes slightly yellow at the top. Bacteria that cannot metabolize the sugar will either produce no color change or will turn the broth blue. Two organisms were used for this exercise: Escherichia coli (Ec) and Pseudomonas fluorescens (Pf). One of the pairs will have a mineral oil overlay added to cultivate an anaerobic environment and force fermentation. If the sealed broth is green or blue and the unsealed is yellow, then oxidation has taken place; if the sealed broth is yellow throughout and the unsealed is also yellow throughout, then the organisms performed oxidation and fermentation or fermentation only; if the sealed medium is slightly yellow at the top and the unsealed is slightly yellow at the top, then the bacteria has performed oxidation and slow fermentation or slow fermentation only; finally, is the sealed tube is green or blue and the unsealed tube is also green or blue, then the bacteria has no sugar metabolism and is nonsaccharolytic. Exercise 5-3 The phenol red (PR) broth is a differential test made as a base in which a carbohydrate is added. Peptone and the pH indicator phenol red is added to the base medium. Phenol red is yellow below pH 6.8, pink above 7.4, and red between. Before given to students, the medium is

Aseng 9 prepared at a pH of 7.3 so it is red. An inverted Durham tube is added to each tube as an indicator of gas production. Acid production as a result of fermentation of the carbohydrate lowers the pH below 7.0 and turns the broth yellow. Deamination of amino acids given by the peptone results in an alkaline reaction from the ammonia that is produced, which increases the pH and turns the medium pink. Gas production from fermentation is shown by a bubble in the Durham tube. The PR broth is used to discriminate the members of Enterobacteriaceae and to distinguish them from other Gram-negative rods. It can also be used to differentiate between Gram-positive fermenters. Students will be inoculating PR broths with four organisms: Providencia alcafaciens: (Pal), Serretia marcescens (Sm), Eschericia coli (Ec), and Citrobacter freundii (Cf).

Exercise 5-20 SIM medium is used for the determination of three bacterial characteristics: sulfur reduction, indole production from tryptophan, and motility. The medium contains casein and animal tissue as sources of amino acids and sulfur in the form of sodium thiosulfate. Sulfur reduction to H2S can be accomplished by cysteine desulfurase catalyzing the hydrolysis of cysteine to pyruvate or thiosulfate reductase catalyzing the reduction of sulfur to H2S at the end of an anaerobic respiratory electron transport chain. Both of these systems produce hydrogen sulfide gas; in the medium, the H2S combines with iron to form ferric sulfide, a black growth. This black color is an indication of the sulfur reaction and a positive test. Absence of black precipitate indicates no sulfur reduction and a negative test. Indole production was not observed in this exercise. The motility characteristic of the

Aseng 10 bacteria is possible by the reduced agar concentration and the method of inoculation. The medium is stabbed once with a needle; motile bacteria are able to move in the medium and can be seen by the radiating growth from the central stab line: radiates in all directions and appears slightly fuzzy indicates motility . Students stabbed the SIM medium with two organisms: Escherichia coli (Ec) and Citrobacter freundii (Cf). Sulfur reduction are recorded in the table as positive for black precipitates in the medium with bacteria that can reduce sulfur, or negative if there is no black in the medium meaning that sulfur is not reduced. For observations of motility: growth radiating from the stab line means positive motility, no radiating growth signifies that the bacteria is non motile, and blackened media means that motility cannot be determined (Leavall, Sandie, et al, 2007).

Results: Exercise 4-1 Organism

Growth (N/P/G) PEA

Interpretation and Presumptive ID NA

Ec

Poor

Good

(-)

Er

Good

Good

(+)

Ss

Good

Good

(+)

Table 1. Three organisms shown in the table were grown on PEA plates and NA plates for comparison to determine whether the organisms were inhibited by phenylethyl alcohol.

Exercise 4-3 Organism Ll

Color Result Black

Positive/Negative (+)

Interpretation and Presumptive ID ● Good growth

Aseng 11 ● Plate was dark Er

N/A

(+)

● Good growth

Pal

N/A

(-)

● Poor growth

Table 2. BEA produced plates that may have had growths observed as well as color changes in the media. The growth and color both play roles in determining the identification of each organism.

Exercise 4-4 Organism

Growth

MSA Growth Color (Y/R)

TSA

Interpretation and Presumptive ID

NA

Ec

None

Good

Red

Er

None

Good

Red

Sc

Good

Good

Red

Ss

Poor

Good

Yellow

(discussed below)

Table 3. TSA was used instead of MSA but still has the same results. Table shows the growth patterns as well as color changes of organisms on high salt concentration media.

Exercise 4-5

Organism

Growth MAC

MSA Growth Color (R/C)

Interpretation and Presumptive ID

NA

Er

None

Good

Colorless

Positive; no lactose fermentation

Ec

Poor*

Good

Pink/Red

Negative; Lactose fermenter

Pal

Poor*

Good

Colorless

No lactose fermentation

Ka

Good

Good

Pink/red

Negative; Lactose fermentation

Cf

Poor*

Good

Pink

Negative; Lactose fermenter

Table 4. The growth and colors observed on the MacConkey media convey whether the organisms are able to ferment lactose or not. Asterisk indicates the results that did not coincide with the expected results.

Aseng 12 Exercise 4-6 Organism

Growth

EMB Growth Color (Pi/D/C)

EMB

Interpretation and Presumptive ID

NA

Er

Poor

Good

N/A

Ec

Good

Good

Dark

Pal

Good

Good

Dark*

Ka

Good

Good

Pink

Cf

Good

Good

Dark

(discussed below)

Table 5. EMB agar contains dye that inhibit the growth of Gram-positive organisms and react with lactose fermenters who products turn growth dark purple or black. Identification will be based on growth and color changes. Asterisk conveys a mistake in the experiment.

Exercise 5-2

Organism

Color Results Sealed

Symbol

Interpretation

Unsealed

Ec

N/A

Pf

Green

Yellow

O

Oxidation

Table 6. Unfortunately, we did not produce results for Ec. We placed oil in both the sealed and unsealed flask and did not produce results for Ec.

Exercise 5-3

Organism

Results PR Dextrose

Pal

Yellow

PR Lactose Pink

PR Sucrose Pink

Interpretations (expected results) Dext: A/Lac: -/Suc: -/-

Sm

Ye...