Identifying Unknown Microorganisms: Key Testing Methods

docx

School

Gyan Vihar Scholl of Engineering And Technology *

*We aren’t endorsed by this school

Course

Subject

Biology

Date

Nov 24, 2024

Type

docx

Pages

Uploaded by shubhangicp

To identify unknown organism in microbiology we have to conduct several tests for the confirmation of microorganism. There are three major tests are performed for the identification and they are 1. Gram stain 2. Fermentation test / selective media 3. Differential media test The bacteria under investigation are an unknown sample organism. To identify type of organism gram stain test is performed. Gram stain test: Gram stain test: gram stain test is performed with the crystal violet or methylene blue as primary color. The organism that retains primary color and appears purple brown under microscope is gram positive organism. Those appear red under microscope are Gram-negative organism. Gram stain distinguishes between two cell wall organism and thick and thin and this test only provide information gram negative and positive organism. Method: This method is performed by taking a sample of orgamism and putting it in the mixture of potassium salt solution having gram stain and kept for 24 hours. When it has been stained with iodine and sample turns red because gram negative cell has thick cell wall and it resist the binding of stain. Initially all organism take the violet dye when solvent is used the lipid layer from the gram negative organism dissolved and primary stain is lost. In the gram positive solvent dehydrate cell wall with the closure of pores preventing diffusion of violet iodine complex and thus bacteria remain stained. In the final step basic Fuchsin stain is used to give decolorized bacteria pink color so that it can be identified easily. The color of organism of sample is gram negative in nature.

Is this organism's morphology bacillus- or coccus-shaped? What evidence supports your reasoning? The sample given when seen under the microscope morphology of the sample is coccus shaped. Coccus shaped bacteria is a spherical bacterium and the figure shows that the bacterium in the figure are spherical in shape. From the shape of grouping they are in single, group of four or are in cluster or in chain but round in shape. This test identifies general type of bacteria but is known as preliminary test. To confirm exact type of bacteria we are required to conduct further tests. As our sample is coccus in nature we will conduct capsule tests What test do you perform next and why? (Reference the decision tree.) Capsule stain test: To confirm the nature of bacteria capsule stain test is most suitable. Capsule stain test is to reveal the presence of the bacterial capsule. It is difficult to see the bacterial cell by standard simple staining procedure. Many bacteria including both gram – positive and negative may be surrounded with an outer polysaccharide – containing layer that is glococalyx. When this layer is tightly bound with the cell it is called capsule. These polysaccharides layers may contain poly- alcohols and polyamines. These capsules are protective structures and adhere to surface and other bacteria are contributing to bio-film formation. In Capsule stain method a combination of dyes are used i) basic dye that interact with negative ions of the bacterial cell. ii) A mordant that precipitation of the capsular material and they are metal ions, alcohol and acetic acid and iii) an acidic stain used to color the background. At the completion of the preparation, the capsule is revealed as a clear circle of light between the colored background and the stained cells. In the method crystal violate is used as the primary stain, interacting with the protein material in the culture broth or added during the staining and copper sulfate serves as the mordant. At the completion of stain the bacterial cells and the background will be stained by crystal violet while unstained capsules will appear white.

Do you see space around the cells in this image? In this stained method there is no hollow surrounding the cocci-shaped cells means no capsules are present. What is the significance of the space or lack of space? What does it mean to your investigation? If space is present around the Cocci that means capsules are present around the Cocci as no space is visible no capsule is present. Does this organism form a capsule? What evidence supports your reasoning? This organism does not form capsule and as there is no space is present around the cocci and we will conduct the fermentation test to confirm the type of bacteria. Fermentation test : Fermentation test is a metabolic process that some microorganism uses to breakdown glucose and other sugar when oxygen is no available, or could not be used by the microorganism. In the fermentation process bacteria produces ATP to meet their energy requirement. In the fermentation process metabolic reaction produces glycolysis and variety of other products like acid, alcohol, gas. End product of this test is the characteristic of the specific bacterial species. Fermentation is also possible from non-sugar molecules. Fermentation procedure can take place in presence of certain bacteria with unusual compounds like aromatics, glycerol and acetylene. To confirm that our unknown sample is helping the fermentation process or not we have to conduct fermentation test. Fermentation test: In a sample that contains carbohydrate some bacteria produces gases during fermentation process. To detect these gases a Durham tube is used. In this test a small inverted glass tube that

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

is placed with the larger glass tube containing fermentation media. During this test if gases (CO 2 ) are produced during the fermentation process, a bubble will form at the top of the Durham tube. Principle: When microorganism ferment carbohydrate an acid or acid with gas are produced and depend on the type of organism products of bacterial fermentation include lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, carbon dioxide and hydrogen. The production of the acid lowers the pH of the test medium and color of indicator changes accordingly. Color change will occur when a sufficient amount of acid is produced, as bacteria may utilize the peptone producing alkaline by products. When we use phenol red as indicator un-inoculated sample will be orange red while fermented product will be yellow and unfermented product will be pink red. During test Durham tubes are inserted upside down in the test tubes to detect gas production. Of the test organism produce gas, the gas displaces the media present inside the tube and gets trapped producing a visible air bubble. Based on the characteristics reactions observed, bacteria can be classified as: Fermented with acid production only Fermented with acid and gas production Non-fermented Test procedure : Phenol red carbohydrate broth is used in carbohydrate fermentation test. The carbohydrate source can be glucose broth with phenol red. Composition of the media: Take specific phenol red carbohydrate test media Media contain following ingredients TRipticase or protease peptone No. 3 10 g Sodium chloride 5 g Beef extract 1 g Phenol red 7.2 ml of 0.25% phenol red solution or 0.018 g Carbohydrate source glucose 10 g

Preparation of the media: Prepare broth media by mixing all ingredients in 1000 ml of distilled water and heating it gently to dissolve all the ingredients. Fill 13 x 100 mm test tubes with 4 – 5 ml of phenol red glucose carbohydrate broth. Insert a Durham tube to detect gas production Autoclave the prepared test media to sterilize at 121deg. C for 3 min The prepared broth media will be a light red color and the final pH should be 7.4+/- 0.2 Inoculation and incubation: Aseptically inoculate each test tube with the test microorganism using inoculating needle or loop. Incubate tube at 35 to 37Deg.C for 24 hours. After the incubation the liquid in the tube turns yellow. It indicates that there is drop in the pH because of production of acid because of fermentation of the carbohydrate present in the media. If the tube containing medium remains red then the microorganism is not fermenting the glucose present in the media. Gas production: A bubble in the small Durham tube is seen showing that the test is positive for negative test there will be no gas bubble. The given microorganism has converted the solution to yellow What color is the tube? Is gas produced? Color in the tube is yellow and there is a small bubble in the tube confirming that fermentation is taking place and test is positive. Are the results positive or negative? What does that mean for this organism? The result of the test is positive and that shows that the microorganism is capable of converting higher molecules of organic compound in the lower molecular weight compounds generating carbon dioxide that is collected in the Durham tube. Catalase Test: T his test is used to identify organism that produce enzyme catalase. The enzyme produced detoxified hydrogen peroxide by breaking it in to water and oxygen gas.

Catalase 2 H 2 O 2 2 H 2 O + O 2 The bubbles resulting from production of oxygen gas clearly indicates a catalase positive result. Bacteria that are aerobic in nature conduct aerobic metabolism, produces hydrogen peroxide as toxic byproduct of their metabolism and cause intracellular damage, such as catalase. Only species that have catalase genes will make catalse, will test, catalase test positive and can be identified by this test. This is a simple test. In this test a sample specimen is placed on the slide and hydrogen perocxide is placed on it. If catalase is present hydrogen peroxide will break into water and oxygen and will produce water and oxygen. This test does not require any special type of medium or organism that have been grown on blood agar. This is because there is a catalase activity in blood that would produce false results. The anaerobic bacterial species does not have catalase cannot survive in aerobic conditions. Method: 1. Take a glass slide and bottle of hydrogen peroxide 2. Take a sterilized inoculated loop or needle, and add smear a small amount of bacteria onto the dry slide. 3. 3. Place a drop of Hydrogen peroxide on top of the bacteria. 4. Observe that bubbles are produced or not on the top of bacteria. Result: Bubbles produced – Catalase positive confirming that our unknown microorganism is aerobic in nature. What evidence supports your reasoning? The color of phenol red is converted to yellow indicates that the test is positive and a bubble in the Durham tube also confirm that the microorganism is producing gas and participating in fermentation. This confirms that the given microorganism is Neisseria Gonnorrhoeae. As per the decision tree this organism is cinfirming the following tests:

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

S. No. Test performed Results of the tests Remarks 1. Test Gram stain reaction Gram stain morphology Descriptio n Interpretatio n pink Gram negative Round and paired Coccus Capsule stain test No capsule observed 2 Fermentation test Glucose Yellow bubble Test positive This confirm as per decision tree that microorganism is Neisseria gonorrhoeae Maltose Negative Pink in color sucrose Negative Pink in color catalase test Hydrogen peroxide Bubbles are formed during test within two to three second Positive test confirming unknown microorganism is catalse positive Neisseria gonorrhoeae, also known as gonococcus is a species of Gram negative diplococcic bacteria Kirby-Bauer diffusion test: Next test we will perform will be Kirby-Bauer Diffusion Test: Purpose: The purpose of the Kirby Bauer disk diffusion susceptibility test is to determine the sensitivity or resistance of pathogenic aerobic and facultative anaerobic bacteria to antimicrobial compounds. The microorganism is grown on the Mueller Hinton Agar in the presence of various antimicrobial impregnated filter paper disks. the presence or absences of the ability of that compound to inhibit that organism. Theory: Determination of bacterial resistance to antimicrobials is an important part in the identification of resistance of drug of any microorganism. In the disk diffusion method of Kirby and Bauer test a 6 mm filter paper disk impregnated with a known concentration of an antimicrobial compound is placed on a Mueller Hinton agar plate. Water from the agar will be absorbed by disk and antimicrobial begins to diffuse into the surrounding agar. The rate of diffusion through the agar is not as rapid as the rate of extraction of the antimicrobial is highest out of disk. Therefore the concentration of antimicrobial is highest close to the disk and logarithmic reduction in

concentration occurs as the distance from the disk increases. The rate of diffusion of antimicrobial through the agar is dependent on the diffusion and solubility properties of the drug in MH agar and molecular weight of the antimicrobial compound. Larger molecular weight compounds will diffuse at slower rate than lower molecular weight compound. These factors in combination; result in each antimicrobial having a unique breakpoint zone size. This indicate susceptibility to that antimicrobial compound. If agar plate has been inoculate with a suspension with a suspension of the pathogen to be tested prior to the placing of disk on the agar surface, simultaneous growth of the bacteria and diffusion of the antimicrobial compound occurs. Growth occurs in the presence of an antimicrobial observed zone of inhibition measurement results indicate that the organism from the part E is resistant, susceptible or intermediate susceptible, to each antibiotic test. Compound when the bacterium reach a critical mass and can overpower the inhibitory effects of the antimicrobial compound in test hours of 4 to 10 hours and is a characteristic of each species, and influenced by the media and incubation temperature. The size of zone of inhibition of growth is influenced by the depth of the agar as the antimicrobial diffuses in three damnations. Thus a shallow depth of agar will produce larger zone compare to the deeper layer. The point at which critical mass is reached is demonstrated by a sharply marinated circle of bacterial growth around the disk. The concentration of antimicrobial compound at this margin is called the critical concentration and is approximately equal to the minimum inhibitory concentration obtained in broth dilution susceptibility tests. RECIPE Sterile saline in 2-ml tubes 1 0.5 McFarland standard 18- to 24-ho Vortex 2 Wickerham card Sterile swabs 3 Mueller-Hinton agar plates, 100 mm or 150 mm Inoculating lo 4 Caliper or ruler Bact-cinerato 5 Antibiotic disksb Alcohol pads 6 Forceps 35°C to 37°C

Method: 1. Take a sterile swab into the broth and any excess moisture is removed by pressing swab against the test tube wall. 2. Swab the surface of Agar completely and after complete swabbing the plate turn it 90Deg and repeat the swabbing process. 3. Run the swab around the circumference of the plate before discarding it in the discard bag. 4. Allow the surface to dry for about 5 Minutes before placing antibiotic disk on the agar with the help of forceps. Remove antibiotic disc from the dispenser with the help of sterilized forceps. 5. Lightly touch the each disc with sterilized inoculate loop to make sure that it is in good contact with the agar surface and incubate at 37DegC. Interpretation: Place the mm ruler across the zone of inhibition, at the widest diameter. Now measure edge to edge of the zone to the other edge If no zone it is zero and if we observe some distance than report it sensitive resistant or intermediate. Interpretation :

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Observation Table: Resistant Intermediate Susceptible Amoxicillin 42 mm Ciprofloxacin 42 mm Doxycycline 42 mm Conclusion: 1. The unknown microorganism is identified as Neisseria gonorrhoeae 2. The microorganism is confirmed by catalase test 3. 3. The microorganism is showing intermediate nature with Amoxicillin and is susceptible to ciprofloxacin and Doxycycline.

References: https://asm.org/Protocols/Catalase-Test-Protocol https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Microb iology_Labs_I/09%3A_Kirby-Bauer_(Antibiotic_Sensitivity)#:~:text=In%20Kirby%2DBauer %20testing%2C%20bacteria,the%20antibiotic%20inhibits%20bacterial%20growth .

Related Documents