StudySheetMBG353

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Jan 9, 2024

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Name of Media Test For Organism Observation Of Media Results/Conclusion Lactose Broth Lactose 1 Still Red, No Gas formation (-/-), fermentation -ve 2 Red to yellow color, Bubble trapped (+/+), fermentation +ve Glucose Broth Glucose 1 Still Red, no gas formation (-/-) fermentation -ve 2 Red to Yellow Colour Bubble trapped (+/+) fermentation +ve Starch Agar Plate Presence of amylase 1 Clear Zone Around Growth (+,starch hydrolyzed + ve amylase) 2 Blue Colour Formed around growth -,starch not hydrolyzed -ve amylase Urea Slant Presence Of urease 1 Bright Pinkish purple on slant +, urease +ve 2 No colour change still yellow -,urease -ve Tryptophan Broth Test for Indole 1 No change, still work - 2 Red Ring Formed on top + MR Broth Mixed Acid Fermen tation 1 No Red formation, still Clear - 2 Red to Light Red colour + VP Broth Acetyl Methyl Carbinol presence 1 Light Red colour formed + 2 No colour change, still clear - Citrate Citrate 1 Bright blue color +

Slant growth on slant 2 No colour change, Still Dark Blue -

2. The IMVIC pattern for the bacterium E.coli as follows: ++-- In the same manner, write down the IMVIX patterns for organisms #1 and #2 from the results in your table: #1 - -++ #2 ++- - 1. What is the function of Iodine in the starch hydrolysis test? The function of Iodine is to react with starch to form a dark blue-colored complex. If the area is clear, that means the organism broke down the starch that its due to its production of amylase. 2. What is the function of the Durham tube in the carbohydrate broth? The function of the Durham tube is to detect the gas production. Gas will be trapped in Durham tube and a bubble will form. The presence of a bubble indicates fermentation and gas production of fermentation. 3. a) Both the MR and VP tests determine whether a bacteria can use an organic compound as a source of energy. What is this compound? b) Even though the energy source is the same, the MR and VP tests determine separate metabolic. What are the end products of these pathways for each test? For the MR, it will be mixed stable acids (lactic, acetic, formic acid). For the VP, it will be acetyl methyl carbinol. 4. How is indole production detected in the indole test? The presence of indole is present when a red ring is present at the surface of the broth. The presence of Indole when a red ring is formed at the top of the broth. Starch Agar Plate: Testing for Amylase Typtophan Broth: Test for Tryptophan broth transforms to Indole via the addition of kovac reagent MRVP Broth

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Mixed Acid Fermentation Metabolizes acids to other acids, from glucose Lactic acid, Formic acid, etc VP Acetyl methyl, carbinol from glucose Metabolizes pyruvic acid to other acids (ie. lactic, formic, and acetic acid), from glucose Lactate, Formic, etc VP Neutral products like Acetyl methyl carbinol and ethyl alcohol from glucose metabolism Interpretation of Results Record your results in the table. Follow the interpretation guidelines below. Carbohydrate Fermentation Positive test for fermentation = acid production (yellow colour). You must also record whether or not gas is produced. Do this by checking the inverted tube for the presence of a “good size” air bubble. Negative test for fermentation = original red/orange colour (no acid has been produced). Indicate your results as acid/gas. Choose between: + / + (acid & gas), + / - (acid but no gas), or - / - (no acid or gas) Starch Hydrolysis After incubation, place several drops of Gram’s iodine onto the bacterial cultures. Try to cover both the culture and the surrounding media – ie. aim for an ‘edge’. Examine the plate for a clear zone surrounding the growth --- a positive result. Negative result? – the media immediately surrounding the bacterial growth appears deep blue.

Urea Hydrolysis Following incubation, observe the tubes for growth and any colour changes. Positive reaction: a deep pink-purple colour. IMViC Tests For the indole test, add about 5 drops of Kovac’s reagent directly to the tubes of tryptone broth. Do not shake the tube. Observe the tube immediately for the formation of a bright red ring at the top of the broth (positive). If the top portion remains yellowish or brownish, this is a negative result. For the MR-VP tests, aseptically transfer 1 ml of each of the cultures to two separate sterile plastic tubes. One plastic tube will be used for the MR test, the other plastic tube will be used for the VP test. Add the following test reagents to the tubes as indicated: MR test: Add 3 drops of the methyl red reagent to one tube. Positive result = red colour (indicates acid production) VP test: Add 15 drops of Barritt’s reagent A, mix well. Then add 5 drops of Barritt’s reagent B, mix well. Let tube sit for 10-15 minutes, shaking often. Positive result = light red colour For the citrate test, observe the tube for growth and colour changes. Positive result = growth + deep blue colour, Negative result = no growth, media remains green

True motility True motility (self-propulsion) may be due to structural appendages called flagella (singular, flagellum). These are thin protein tubes attached to the bacterial cell wall which rotate to provide propulsion for the cell. Not all bacteria have flagella; hence, some types of bacteria are non-motile. Flagella are generally too thin to be seen in live, unstained bacteria even at high magnification. While it is not possible to see flagella using the light microscope, true motility can be detected in live bacteria by observing rapid changes in cell position. True motility will result in the bacteria spinning around (rotational movement) or traveling long distances across the field of view (translational movement), while changing directions from time to time. Brownian motion and streaming All microbes suspended in liquid medium will appear to move quickly back and forth. This type of movement is called Brownian motion and is due to the random vibrations produced when the microbes collide with water molecules. Brownian motion should not be confused with true motility, as even non- motile organisms will display this type of behavior. In this exercise you will examine live bacteria for motility using two techniques: the wet mount and the hanging drop mount. Motility Media Motility media is a semi-solid growth media that is used to determine bacterial motility. It is an example of an indirect method of motility testing. In addition to basic nutrients motility media also contains: • 0.5 % agar – Routine solid media usually contains 1.5 % agar. The lower concentration of agar in the motility media allows the bacteria to migrate away from the line of inoculation. The presence of agar prevents diffusion of the cells, so that any distribution of bacteria throughout the media results only from true motility. triphenyl tetrazolium (TPT) – tetrazolium salts are used as an indicator of bacterial growth. During growth, bacteria are able to take up and reduce the tetrazolium, producing an insoluble, red compound called formazan . In this way, the site of bacterial growth in the tube can be easily identified by the presence of a dark red color.

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Hanging Drop Mount 1. Place a small drop of bacterial culture in the middle of a cover slip. 2. Using a toothpick spread a small amount of petroleum jelly around the outside of the depression on a cavity slide. 3. Place the cavity slide over the drop of culture so that the cavity is covering the drop, but not touching the drop. Gently press down to form a seal. 4. Carefully invert the slide so that the cover slip is now on the top and the drop of culture is hanging in the depression. 5. Follow the steps below for focusing on the bacteria. Figure 1 : Top view of cavity slide with petroleum jelly distributed in thin lines around the depression (shown as a circle). Figure 2: Method for placing the the cavity slide to capture the drop of culture inside the depression. Note that the drop does not touch the cavity slide and maintains a convex shape.

Wet mount 1. Place a single drop of culture on a clean, labelled slide. 2. Cover with a cover slip. 3. Follow the steps below for focusing on the bacteria. Viewing Live Cells Under the Microscope 1. Close the condenser diaphragm 90%. 2. Begin with the 4X objective, then switch to 10X and finally 40X. Never use the oil immersion lens to view live cells. Why? 3. For the wet mounts, first focus on the edge of an air-bubble, or on the edge of the cover slip. This edge will appear as a solid black line. For the hanging- drop mount, first focus on the edge of the drop. The cells should now also be in focus. 4. Under 40X, look for small black circles or rods in the liquid. If you are focused correctly and still cannot see the cells, try closing the diaphragm even more or lowering the condenser in order to increase contrast. 5. Note the shape and movement of the cells. Determine whether your organisms show true motility or Brownian motion. Interpretation of Results Complete the provided worksheet for the organisms being examined. Motility Media: Notice the way the media has changed colour in the presence of growing bacteria. You may notice that the bacteria have only grown in the vicinity of the path that was created by your inoculating needle. What does this indicate? Otherwise you might notice that the entire tube has changed colour. What does this indicate?

The Endospore Stain Introduction Certain bacteria are able to form specialized structures called endospores . These structures can resist a variety of chemical treatments, extremes of temperature, and dry conditions. As such, they represent a type of survival mechanism for the cell. (Note that spores are killed, however, by autoclaving which is why we autoclave all contaminated materials in the laboratory). Clostridium and Bacillus are two genera which commonly produce these structures. While what “triggers” vegetative cells to form endospores is not completely understood, it appears to occur when the cells are stressed due to lack of nutrients or water or when exposed to extreme environments. The endospore staining technique is an example of a structural stain which is designed to better allow the microscopic visualization of these structures. Two stains are used; Malachite green (which stains the spores) and safranin (the counterstain which stains vegetative cells and sporangia). Heat (steam) is used to enable the malachite green to penetrate the thick spore coat. Procedure 1. Work in Pairs 2. Obtain a plate containing the bacillus cultures. These will be shared between you and your partner 3. Prepare air-dried and heat-fixed smears of the two cultures. 4. Fill a staining tray three-quarters with water and place on a hot plate. 5. Heat the water to create a “steam bath”. 6. Place small squares of paper toweling over the prepared bacterial smears and place the slides on the wire portion of the staining tray. 7. Immediately saturate the paper toweling with 5% malachite green stain. 8. Do not let the stain “dry out”! Continue to add the malachite green as necessary. Stain the preparations for 10 minutes.

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9. Remove the staining tray from the hot plate and allow the slide to cool. 10. Remove the paper toweling from the slides. 11. Rinse the slides with water, then counterstain with safranin for approximately 1 minute. 12. Rinse the slides with water and pat dry with bibulous paper. 13. Examine the slides using the microscope and the oil immersion technique. Interpretation of Results Note that the spores may be detected within the cell (sporangium) in “younger” cultures (at early stages of sporulation) or may appear alone (ie. outside the vegetative cells) in “older” cultures (at later stages of sporulation). If the spores are found within the cells, make a note of their position. This can be a taxonomic feature; ie. may aid in the identification of the organism. a. central b. terminal c. sub-terminal d. terminal with swollen sporangium e. extruded

Introduction Disinfectants are chemical formulations that reduce the number of bacteria to a safe level. Disinfectants do not sterilize, and are generally ineffective against bacterial spores. The effectiveness of a disinfectant depends upon the concentration of the chemical, the length of time of treatment, the number of bacterial cells initially present and the susceptibility of the test organism. Common disinfectants include 70% alcohol, chlorine bleach, soaps and detergents. Antiseptics are compounds that are used to disinfect living tissues. 3% hydrogen peroxide, iodine, and 70% alcohol can be safely used as antiseptics. Additionally, many (human) cultures have a knowledge and use of compounds that can be used to treat infections or clean surfaces. Frequently, the scientific and medical community “discovers” substances have been known for centuries in some cultures. Often these “home-remedies” work due to trace compounds that are naturally found in these substances. The disc-diffusion assay is a method used to determine the efficacy of a compound to destroy a test organism. It can be used to compare several different concentrations of a disinfectant, or directly compare several types of disinfectant against one type of microbe. In this lab, you will investigate household disinfectants, and also a product you find at home to treat bacteria/infections and bring a sample to lab. You will make a liquid preparation of the compound and describe it to the class. Does this substance have historical or cultural use? Please share. You will compare your compound with other common household disinfectants. Do not bring illegal substances, explosives, or otherwise dangerous or noxious substances into school. Use common sense. If handling the compound requires special PPE, handling, or equipment, do not bring it in . If you’re unsure, please ask your instructor for approval.

The disc-diffusion assay: In this exercise, we will spread a “lawn” of bacteria on an agar plate to achieve confluent growth. Sterile paper discs will be applied using sterile forceps. We will apply household disinfectants and our home-remedy on these discs. During incubation, the disinfectants will diffuse out of the paper disks into the surrounding medium forming concentration gradients around the disks. Bacteria that are sensitive to a particular concentration of a compound will not be able to grow within this area, which is called the zone of inhibition. Most household products have proprietary formulations, and we do not know exactly how much of the agent is in each product. Following incubation, the diameters of these zones of inhibition can be compared to determine the relative sensitivity of the organism to the substance. Figure 1: Arrangement of paper discs on the agar. Distribute your paper discs on to the agar in a pattern similar to the one shown above. Prior to incubation, there will be no growth visible (left). After incubation, you will see zones of inhibition of varying sizes (right). More effective disinfectants will have larger zones.

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Figure 2 : The zone of inhibition is measured with a ruler or calipers. Zone sizes can be recorded for several compounds on one plate and compared. Larger zones have greater inhibitory effect against the bacteria spread on the plate. Image from https://www.biologycorner.com/worksheets/case_study_bacteria_resistance.html Antibiotic Sensitivity Testing Antibiotics are chemical compounds produced by a microorganism which kill or inhibit the growth of other types of microorganisms. In nature, bacteria and fungi produce antibiotics as defensive mechanisms against competing microbes. Antibiotics can be purified from microbes and are used as powerful medicines to combat bacterial infections. Penicillin was the first antibiotic discovered and remains the most widely used antibiotic today. This antibiotic is produced by the mould, Penicillium. Penicillin kills bacteria by attacking the protective peptidoglycan cell wall that surrounds a bacterial cell. Other antibiotics, such as erythromycin and tetracycline attack bacteria by blocking protein synthesis. A broad spectrum antibiotic is one that is capable of killing both Gram- positive and Gram-negative cells. Antibiotics are extremely effective medicines because they demonstrate selective toxicity. Selective toxicity means that the antibiotic will attack and kill bacterial cells without harming human cells, by targeting structural and molecular differences between bacterial and human cells. In this exercise you will determine the sensitivity of Gram-positive and Gram-negative bacteria to several common therapeutic antibiotics using the Kirby-Bauer disc diffusion assay. In this assay, the test bacteria are spread evenly over the surface of a Mueller- Hinton agar plate. Mueller-Hinton agar is the standard media used in antibiotic sensitivity testing because it supports the growth of most pathogens and is low in certain antibiotic inhibitors. Following inoculation, paper discs containing specified dosages of various antibiotics are placed on the surface of the plate and the plate is incubated at the appropriate growth temperature. During incubation, the antibiotics diffuse out of the paper disks into the surrounding medium forming concentration gradients around the disks. Bacteria that are sensitive to

a particular concentration of an antibiotic will not be able to grow within this area, called the zone of inhibition. Following incubation, the diameters of these zones of inhibition are measured and compared to standards to determine the sensitivity of the organism to the antibiotics. Figure 1 : On this incubated agar plate, bacteria has been spread uniformly across the entire surface, creating what is sometimes referred to as a “lawn” (bacterial growth, bacteria marked as “a”). Small discs of sterile paper have been applied to the surface (discs marked as “b”) . The discs were dosed with a known quantity of antibiotic, or a disinfectant solution. The antimicrobial compound diffused out of the paper, creating a zone of inhibition where the bacteria could not grow (c).

Use-Dilution Assay Introduction As we learned this week in lecture, the Use-Dilution assay is used to determine the effective concentration of a disinfectant compound on a simulated surface. In this lab we will use one of the disinfectants and one of the bacterial species from our disc-based assay last week and determine the appropriate concentration to use it at. The AOAC Use-dilution method is an assay used to determine the efficacy of a liquid disinfectant on an inanimate surface. In this test, objects called carriers are “exposed” by submerging in fresh solution of bacteria. In a standard assay, Staphylococcus aureus (Gram positive, pathogenic), Salmonella enterica (Gram negative, pathogenic) and Pseudomonas aeruginosa (Gram negative, pathogenic) are used to coat the carriers. These carriers are submerged in disinfectant solution for a defined time, and then subsequently placed into tubes of non-selective growth media and incubated. To read the result, the technician will examine the tubes of media for signs of growth. A tube that has growth after incubation is considered a positive result, which indicates that this treatment did not inhibit or destroy the bacteria. A full-scale use-dilution assay will typically use 60 carriers, and the AOAC recommends the following as a passing criteria for a disinfectant:  For each microbe, three complete and separate replicated trials should be done  For S. aureus , 0-3 positive carriers is a passing result  For P. aeruginosa , 0-6 positive carriers is a passing result.  For S. enterica , 0-2 positive carriers is a passing result.

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a) b) c) Figure 1: Simplified illustration of the Use-dilution assay. Carriers (penicylinders) are submerged in bacterial culture, removed, and allowed to dry. (a) Note that the carriers should remain upright and not laying down as depicted here. The carriers are then submerged in various dilutions of the disinfecant (b). After the required time, the carriers are moved into sterile broth and incubated (c). In our lab, we will perform a simplified version of the assay. We omit several steps and experimental controls from official method, but overall process remains the same. We will not be using pathogenic bacteria which are reccommended by the AOAC method. Interpretation of Results After incubation, observe the tubes for growth and record your results. Tubes that remain clear demonstrate that the concentration was effective at either inhibiting or destroying the bacteria. The tube with the most dilute concentration of disinfectant indicates, roughly, the Minimum Inhibitory Concentration (MIC) of the disinfectant against the bacteria used in this study . For any tubes that remained clear (no growth), there could still be viable cells that are inhibited, but not killed. The Minimum Bactericidal Concentration is the concentration that kills 99.9% of cells. Consider: Is it possible to experimentally determine the MBC with the results of this experiment? 1. [2 marks] Bacterial cultures grown in liquid broths often have characteristic patterns of growth. Name and describe any two patterns of growth that might be observed in a test tube of inoculated media.

One pattern of growth is turbid where the broth in the test tube appears to be cloudy Another pattern of growth is Flocculent where prescence of large particles are dispersed throughout the broth. Sediment is when prescence of solid material at the bottom of te tube; may swirl up when tube is gently tapped. Pellicle is when thick or thin film covering the surface of the broth. Ring of growth forms at the surface of the broth. Turbid (adj) Flocculent (adj) Sediment (noun) Pellicle (noun) Ring(noun )

2. [9 marks] Using the EXPECTED results from your three organisms and your lab notebook, fill in the following table. If no result exists, write “NA”. EC SEP BS/BT Did the organism grow on MSA? (Y/N) N Y Y/N Provide colour of growth on MSA, if present. N/A Clear Colourless Opaque Beige matte colour/ N/A Did the organism grow on MAC? (Y/N) Y N N Provide colour of growth on MAC, if N/A N/A

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present. Purple/Yellow Did the organism grow on blood agar? (Y/N) Y Y Y Provide name of hemolysis reaction if growth was present. Beta Hemolysis Beta Hemolysis Alpha/Gamma Hemolysis 3. [11 marks] Determine if the following statements are true or false and circle your answer. T F Aseptically transferring a loopful of nutrient broth to nutrient broth was used as a negative control. T F MSA detects the ability of Gram-positive organisms to ferment the sugar arabinose. T F The use of a Bunsen burner creates a sterile environment surrounding Bunsen burner flame. T F Differential media limits the ability of organisms to grow allowing the technician to determine if the organism is healthy. T F We should always use the red portion of the flame when using a Bunsen burner as this area is “red hot”. T F In the selective/deiffential media lab, Nutrient agar was used as a positive control for viability of the microorganisms used. T F An ocular micrometer is used to measure cells and needs to be calibrated before use. T F Phenol red is the pH indicator used in MacConkey Agar T F Gram negative organisms can not retain the primary stain during gram staining due to their thin cell membrane. T F Isopropanol is used as the decolorizer during gram staining. T F Gram positive cells appear pink in colour after the gram stain is complete. Gram Negative Stains will absorb everything, but the crystal violet and Iodine treatment since it will be washed away by the Ethanol treatment. It will be unable to do so; because E.coli bacteria is not acidic.

Special Mention: The main use of Bunsen burner is not to produce an aseptic environment, but to prevent upward flowing particles that could contain contaminants or other types of bacteria from falling back into the sterile environment, that the Bunsen burner created. Bunsen burner “locks” the sterile environment. Total Magnification 1000x Draw student’s observation Gram Stain Result Shape / Arrangement Size (µm)