Lab Final – Microbiology – Flashcards
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| What type of media is used for a carbohydrate fermentation test? |
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| PRB Base with various sugars |
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| What does the carbohydrate fermentation test for? |
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| The organism's ability to use the test carbohydrates as a carbon source. |
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| What is the theory of the carbohydrate fermentation test? |
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| Various carbohydrates are incorporated in the media, usually at a 1% concentration. As the carbohydrate is fermented, acidic end products are produced, lowering the pH of the media. This is detected by the pH indicator phenol red changing from red to yellow. An inverted Durham tube present in the test tube traps any CO2 produced as a result of fermentation. |
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| How do you inoculate the media for the carbohydrate fermentation test? |
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| Inoculate as you would for any other broth. |
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| How long do you incubate the carbohydrate fermentation test for? |
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| 18-24 hours |
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| How do you interpret the results of the carbohydrate fermentation test? |
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| Media Yellow: Positive for carbohydrate fermentation Media Red: Negative for carbohydrate fermentation Bubble in durham tube: Positive for gas production No bubble in durham tube: Negative for gas production |
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| Distinguish between respiration and fermentation |
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| Respiration is the process by which organisms use oxygen to supply their tissues and organs and to carry away CO2 to produce energy that the organism needs to survive and fermentation is an anaerobic breakdown of an energy rich compound to supply energy. |
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| Do all microorganisms use pyruvic acid in carbohydrate fermentation the same way? |
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| No, they metabolize it in different ways, thus they produce different end products. |
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| Describe a pathway used for the degradation of carbohydrates by strict anaerobes |
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| glucose + ATP -->glucose-6-phosphate + ADP --> fructose-6-phosphate + ATP --> Fructose-1,6-diphosphate + ADP --> 2 phosphoglyceraldehyde --> 2 Diphosphoglyceric acid + 2 ADP --> 2 ATP + 2 Phosphoglyceric acid+ 2ADP --> Pyruvic acid +2ATP |
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| What kind of media is used for the Triple Sugar Iron Test (TSI)? |
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| A TSI slant |
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| What does the TSI test test for? |
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| the ability to ferment dextrose, lactose, and sucrose and to produce sulfides. |
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| What is the theory of the TSI test? |
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| TSI is a differential medium for Gram-negative enteric organisms. It differentiates organisms based on their ability to ferment the lactose and or sucrose present in the media. (Note: all Gram-negative enteric organisms will ferment the glucose.) The TSI media contains disaccharides lactose and sucrose in concentrations of 1% and glucose at a concentration of 0.1%. The lower concentration of glucose allows for the detection of utilization of this substrate alone. Since glucose is a monosaccharide, it will be used first. The small amount of acid produced in the slant of the tube during glucose fermentation is oxidized rapidly, causing the medium to remain red or revert to an alkaline pH. In contrast, the acid reaction (yellow) is maintained in the butt of the tube because it is under lower oxygen tension. After the depletion of the limited glucose, organisms able to do so will begin to utilize the lactose and or sucrose resulting in an acid slant and butt. H2S production (indicated by the blackening in the butt of the tube) is detected by the sodium thiosulfate in the media, which is reduced by some organisms to hydrogen sulfide, which then reacts with ferrous sulfate in the media yielding the typical black iron sulfide. Cracked or elevated media is the result of production of CO2 from the fermentation of sugars. |
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| How is media inoculated for the TSI test? |
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| stab the butt of the tube and streak the surface of the slant |
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| How long do you incubate the TSI test? |
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| 18-24 hours. Incubate no longer than 24 hours because of the acid reaction in the slant may revert to an alkaline reaction. This is called reversion. |
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| How do you interpret the results of the TSI test? |
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| Red slant/yellow butt (K/A) = glucose fermentation only Yellow Slant/yellow butt (A/A) = glucose, lactose and/or sucrose fermentation Red slant/red butt (K/NC) = no carbohydrate fermentation hydrogen sulfide production results in a black precipitate in the butt. CO2 production is indicated by cracking or elevation of the media. NOTE: some hydrogen sulfide producing organisms may produce so much of the black precipitate, ferrous sulfide, that the acidity produced in the butt is completely masked. However, H2S is reduces, an acid condition does exist in the butt even if not observable and should be recoded as such. |
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| What is the purpose of the TSI test? |
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| To differentiate among the different groups of Enterobacteriaceae which are all gram negative bacilli capable of fermenting glucose with the production of acid and to distinguish from other gram negative intestinal bacilli. |
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| Explain why the TSI medium contains a lower concentration of glucose than of lactose and sucrose. |
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| Permits detection of the utilization of the substrate only. |
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| Explain the purpose of the phenol red in the medium for the TSI test. |
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| To detect the fermentation of carbohydrates by change of the medium from an orange-red to a yellow color. |
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| Explain the purpose of thiosulfate in the medium for the TSI test. |
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| Changes the color in the butt to a black precipitate if H2S is present. |
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| Explain why the test observations must be made between 18 and 24 hours after inoculation for the TSI test. |
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| To make sure that carbohydrates are not depleted so the color change will still be present when the observations are made. |
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| What are the IMViC tests used for? |
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| differentiating members of Enterobacteriacae. |
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| What kind of media is used for the indole production test? |
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| SIM media |
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| What enzyme is responsible for the indole production test? |
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| Tryptophanase |
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| What does the indole test test for? |
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| The organism's ability to produce the enzyme tryptophanase |
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| What is the theory of the indole test? |
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| SIM media contains casein peptone that is rich in tryptophan. If the organism produces the enzyme tryptophanase the tryptophan is broken down into indole, pyruvic acid, and ammonia. |
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| How do you inoculate the media for the indole test? |
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| inoculate with a single stab using a straight needle. Stab in the center of the media to a depth of about 2/3rds of the medium. Make sure to withdraw the needle along the original stab. |
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| How long do you incubate the indole test? |
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| 18-24 hours |
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| What reagents do you add to the indole test? |
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| 5 drops of Kovac's reagent |
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| How do you interpret the results of the indole test? |
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| Blackening of the media: Hydrogen sulfide production Cherry red color in the Kovac's reagent layer: Positive for indole No color in the Kovac's reagent layer: Negative for indole NOTE: Red color will appear in the reagent layer, not in the media. NOTE: Be sure to read hydrogen sulfide and motility before adding reagent. Turbidity of the media: organism is motile No turbidity in the media: organism is non-motile |
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| What type of media is used for the methyl red test? |
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| use MR-VP broth |
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| What does the methyl red test test for? |
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| the organism's ability to produce large amounts of acidic end-products from glucose fermentation. |
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| What is the theory of the methyl red test? |
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| Methyl red positive organisms produce high levels of acid during fermentation of glucose, which can overcome the phosphate buffer system in the media and produce a red color upon the addition of methyl red reagent. |
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| How is methyl red media inoculated? |
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| Inoculate as you would for any other broth |
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| How long do the methyl red tests incubate for? |
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| at least 48 hours. Broth should be turbid after incubation. |
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| What reagents get added to the methyl red test? |
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| 5 drops of methyl red, shake and observe |
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| How do you interpret the results of the methyl red test? |
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| Methyl red is red at pH 4.4 and yellow at pH 6.2 Methyl red following addition of methyl red: Positive Media turns yellow following addition of methyl red: negative Note: MR-VP broth is used for both the MR and VP test NOTE: Use a separate tube for each organism for the MR and one for the VP test. |
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| What media us used for the Voges-Proskauer test (VP test)? |
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| MR-VP Broth |
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| What does the Voges-Proskauer test test for? |
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| The organism's ability to degrade glucose to basic or neutral end products, especially acetylmethylcarbinol. |
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| What is the theory of the Voges-Proskauer test? |
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| The red color produced by the addition of KOH-creatine (Barritt's B reagent) to cultures of certain microbial species is due to the ability of the organisms to produce a neutral end product, acetoin (acetylmethylcarbinol), from the fermentation of glucose. The acetoin is oxidized in the presence of oxygen and KOH (in Barritt's B) to produce diacetyl that reacts with creatine (the other ingredient in Barritt's B reagent) to produce a red color. |
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| How do you inoculate media for the Voges-Proskauer test? |
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| inoculate as you would for any other broth |
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| What reagents do you add for the Voges-Proskauer test? |
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| After incubation, add 5 drops Barritt's A (alpha-naphthol), shake, and immediately add 5 drops Barritt's B (KOH-creatine) located int he refrigerator, shake once. Let sit undisturbed for at least 15 minutes before reading the results. |
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| How do you interpret the Voges-Proskauer test? |
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| Media has red ring at top following addition of both reagents: positive No red color following addition of both reagents: Negative |
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| What type of media is used for the citrate test? |
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| Simmon's citrate slants |
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| What does the citrate test test for? |
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| the organism's ability to use citrate as sole carbon source. |
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| what is the enzyme responsible for the citrate test? |
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| citrase |
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| What is the theory of the citrate test? |
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| Organisms able to utilize sodium citrate as the sole source of carbon will grow on this medium and produce an alkaline reaction as evidenced by a change in the color of the bromothymol blue indicator from green (neutral) to blue (alkaline). Utilization of the citrate releases carbon dioxide that reacts with the sodium and water in the media to produce sodium carbonate (a basic product). This reacts with the pH indicator bromothymol blue to turn the media blue. Organisms able to metabolize the citrate grow. Organisms that cannot metabolize citrate do not grow at all on this medium. |
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| How do you inoculate the media for the citrate test? |
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| inoculate with a needle by stabbing the butt and streaking the slant. |
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| How long do you incubate the citrate test? |
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| 24 to 48 hours |
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| How do you interpret the citrate test? |
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| Slant has growth with intense blue color: positive No growth on slant and media green: Negative |
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| Discuss the medical significance of the IMViC series of tests. |
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| It identifies groups of Enterobacteriaceae that can cause intestinal infections. |
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| Explain the chemical mechanism for detecting indole in a bacterial culture. |
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| Bacterial culture is grown on SIM agar and following incubation Kovac's reagent is added. A positive result shows a red-violet color a negative result turns yellow. |
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| Account for the development of alkalinity in cultures capable of using citrate as their sole carbon source. |
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| The enzyme citrase breakes it down and produces acetate which is then converted to pyruvic acid and CO2. The CO2 combines with sodium and water to form sodium carbonate. |
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| Distinguish between the types of substrates available to cells for H2S production. |
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| organic --> cysteine with enzyme cystine desulfurase inorganic --> Sodium thiosulfate plus bacterial acids |
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| Explain how SIM medium is used to detect motility in the H2S test. |
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| growth radiating up from the stab line indicates that the organism is motile. |
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| Explain the function of the ferrous ammonium sulfate in SIM agar in the H2S test |
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| It's an indicator for H2S production and forms a black precipitate along the stab line. |
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| What type of medium is used for the urease test? |
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| urea broth |
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| What does the urease test test for? |
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| The organism's ability to degrade urea to CO2 + H2O + NH3 |
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| What enzyme is used for the urease test? |
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| urease |
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| What is the theory of the urease test? |
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| When organisms break down urea ammonia is produced creating a basic environment. This is detected by the phenol red incorporated in the media that turns a deep pink color in basic conditions. This test is for identification of Proteus spp. |
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| How do you inoculate the urease test? |
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| inoculate as you would for any other broth |
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| How long do you incubate the urease test for? |
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| 24 to 48 hours |
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| How do you interpret the urease test? |
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| Deep pink color in media: Positive No change in color of media: Negative |
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| Explain the mechanism of urease activity. |
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| Enzyme that attacks the nitrogen carbon bond in amide compounds and forms alkaline end product, ammonia. |
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| Explain the function of phenol red in the urea broth medium for the urease test. |
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| A pH indicator to turn red if environment becomes alkaline indicating that a reaction has take place. |
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| Explain how the urease test is useful for identifying members of the genus Proteus |
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| The members of the genus Proteus reduce urease very quickly where most other organisms do not so it is easier to identify them by this test. |
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| What media do you use for the Nitrate reduction test? |
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| nitrate broth |
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| What does the nitrate reduction test test for? |
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| the reduction of nitrates into nitrites and nitrogen gas. |
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| What is the enzyme responsible for nitrate reduction test? |
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| Nitrate reductase |
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| What is the theory of the nitrate reduction test? |
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| Reduction of nitrate is generally an anaerobic respiration in which an organism derives its oxygen from nitrate. In this test, if the organism reduces he nitrate to nitrite, the nitrite reacts with the reagents sulfanilic acid and alpha-naphthylamine to produce a red color. No color after addition of these reagents indicates nitrates are not reduced or nitrites were reduced further. If the organism possesses a strong nitrate reductase it can further break down nitrites to ammonia or molecular nitrogen. The addition of zinc dust is used to determine if the nitrates were reduced past nitrite stage. If a red color develops after the addition of zinc, the nitrates were not reduced to nitrites. If there is no color after the addition of zinc, the nitrates in the media were reduced beyond nitrites to ammonia or nitrogen gas. |
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| How do you inoculate the nitrate reduction test? |
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| as you would any other broth |
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| How long do you incubate the nitrogen reduction test for? |
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| 24-48 hours |
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| What reagents do you add to the nitrogen reduction test? |
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| 5 drops sulfanilic acid and 5 drops of alpha-napthylamine. Observe for development of red color. If a red color does not develop add a minute quantity of powdered zinc. Observe for the development of a red color. |
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| How do you interpret the results of the nitrogen reduction test? |
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| Red color after adding solutions a and b = reduction of nitrates to nitrites colorless after addition solutions a b and zn = NO3 reduced to ammonia or N2 colorless after addition of solutions A and B and red after the addition of zinc = nitrates were not reduced |
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| Explain the function of the 0.1% agar in the nitrate medium for the nitrate reduction test. |
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| It makes the media semisolid which selects for the organisms that are anaerobic which is required for nitrate reduction |
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| Explain the functions of solutions A and B in the nitrate reduction test. |
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| turn red if nitrate was reduced or stay the same if no nitrate was reduced |
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| If a culture does not undergo a color change on the addition of solutions a and b explain how you would interpret the result of the nitrate reduction test. |
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| 1) Nitrates were not reduced 2) Nitrates were reduced beyond the nitrite stage to ammonia or molecular nitrogen |
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| Explain why the development of a red color on the addition of zinc is a negative nitrate reduction test. |
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| Zinc reduces nitrates to nitrites so if it changes to red when zinc is added that means the original reaction did not take place. |
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| What does the catalase test test for? |
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| The ability of organisms to degrade H2O2 to H2O and O2 |
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| What enzyme is responsible for the catalase test? |
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| catalase or peroxidase |
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| What is the theory of the catalase test? |
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| Hydrogen peroxide and superoxides are produced when aerobes, facultative anaerobes, and microaerophiles use the aerobic respiratory pathway during degradation of carbohydrates for energy production. Accumulation of these substances will result in death of the organism unless they can be enzymatically degraded. Catalase or peroxidase rapidly degrades H2O2 into H2O and O2. All aerobes and some facultative anaerobes are catalase positive. Superoxide dimutase is the enzyme responsible for degradation of the superoxides in catalase negative aerobic organisms. Strict anaerobes are not able to produce these enzymes. Therefore, they cannot be cultivated in the presence of oxygen. |
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| What is the reagent in the catalase test? |
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| Hydrogen peroxide (H2O2) |
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| How do you interpret the results of the catalase test? |
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| bubbles evolved after adding H2O2: Positive No bubbles after adding H2O2: Negative |
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| Explain the toxic effect of O2 on strict anaerobes in the catalase test. |
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| The presence of O2 causes superoxide to form and because the strict anaerobes lack the enzymes superoxide dimutase, catalase, and peroxidase they are unable to break it down. |
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| Illustrate the chemical reaction involved in the degradation of hydrogen peroxide in the presence of catalase. |
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| 2H2O2+ Catalase --> 2H2O + O2 |
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| What does the oxidase test test for? |
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| Cytochrome C |
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| What is the enzyme responsible for the oxidase test? |
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| Cytochrome oxidase |
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| What is the theory of the oxidase test? |
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| Cytochrome oxidase functions in the electron transport system by being a catalyst in the oxidation of a reduced cytochrome by molecular oxygen, resulting in the formation of H2O or H2O2. In the oxidase test, the reagent tetramethyl-p-phenylenediamine dihydrochloride functions as anelectron donor, thereby becoming oxidized to a blackish compound in the presence of the enzyme oxidase and free oxygen. Aerobic bacteria, as well as some facultative anaerobes and microaerophiles, exhibit oxidase activity. |
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| What is the reagent in the oxidase test? |
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| Tetramethyl-p-phenylenediamine dihydrochloride |
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| How do you interpret the results of the oxidase test? |
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| Bacteria black or purple after the addition of reagent: positive bacteria color unchanged after addition of reagent: Negative NOTE: using a loop or a needle for the oxidase test can lead to false positive results due to the oxidation on the wire |
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| What is the function of cytochrome oxidase in the oxidase test? |
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| Catalyze the oxidation of a reduced cytochrome by molecular oxygen resulting in the formation of H2O or H2O2 |
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| Why are strict aerobes oxidase positive? |
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| To keep the ETC functional |
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| The oxidase test is used to differentiate among which groups of bacteria? |
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| Neisseria and Pseudomonas which are oxidase positive enterobacteriaceae that are oxidase negative. |
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| What is the function of the test reagent in the oxidation procedure? |
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| It adds electrons that speed up oxidation and cause a color change from pink to maroon to dark purple |
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| Explain the purpose of the methylene blue dye in the methylene blue reductase test. |
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| It is an indicator. When it loses it's color in an anaerobic environment it is said to be reduced. |
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| Explain why milk sours in the absence of refrigeration. |
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| It sours in the absence of refrigeration because in higher temperatures microorganisms have a faster metabolism and break down the nutrients in milk faster. |
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| Milk also sours, though at a slower rate, while refrigerated. Why? |
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| The microorganisms are still metabolizing the nutrients but at a slower rate. |
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| What is the rationale for selecting E. Coli as the indicator for water potability? |
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| E. Coli is an indicator that water came into contact with fecal matter and many microbes grow due to fecal contamination. |
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| Why is the water analysis experiment qualitative rather than quantitative? |
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| it doesn't measure numbers it measures whether or not there was growth. |
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| Explain why it is of prime importance to analyze water supplies that serve industrialized communities. |
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| because if there is fecal contamination in the water it is likely that there will be disease outbreaks in those areas. |
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| Name one organism that is a gram negative rod. |
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| E. Coli |
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| Name one organism that is a gram positive cocci. |
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| S. Aureus |
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| Name one organism that is a gram positive rod |
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| B. Subtilis |
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| Name one organism that has a A/A -H2S +Gas reaction to the TSI test |
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| E. Coli |
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| Name one organism that has a K/A -H2S -Gas reaction to the TSI test |
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| S. sonnei |
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| Name one organism that has a K/A +H2S +Gas reaction to the TSI test |
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| S. typhimurium |
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| Name one organism that is A/A +H2S +Gas reaction to TSI test |
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| P. vulgaris |
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| Name one organism that is K/NC -H2S -Gas to the TSI test |
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| P. aeruginosa |
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| Name one organism that is motile. |
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| E. Coli |
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| Name one organism that is not motile. |
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| S. Aureus |
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| Name one organism that is + for the indole test. |
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| E. Coli |
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| Name one organism that is - for the indole test. |
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| S. Aureus |
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| Name one organism that is positive for the methyl red test. |
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| E. Coli |
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| Name one organism that is negative for the methyl red test. |
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| E. aerogenes |
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| Name one organism that is positive for the Voges prokastuer test. |
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| E. aerogenes |
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| Name one organism that is negative for the VP test. |
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| E. Coli |
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| Name one organism that is positive for the citrate test. |
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| E. aerogenes |
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| Name one organism that is negative for the citrate test. |
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| E. Coli |
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| Name one organism that is positive for the urea test. |
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| P. vulgaris |
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| Name one organism that is - for the urea test. |
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| E. Coli |
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| Name one organism that is -/+* for the urea test. |
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| K. pneumonia |
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| Name one organism that is positive for the nitrate test |
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| E. Coli |
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| Name one organism that reduces beyond nitrite for the nitrate test. |
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| P. aeruginosa |
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| Name one organism that has no reaction to the nitrate test. |
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| A. faecalis |
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| What is one organism that is positive for the catalase test? |
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| E. Coli |
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| What is one organism that is negative for the catalase test? |
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| E. faecalis |
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| What is one organism that is positive for the oxidase test? |
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| P. aeruginosa |
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| What is one organism that is negative for the oxidase test? |
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| E. Coli |
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| What are EMB plates used for? |
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| differential and selective. Used for the isolation of gram negative enteric bacteria. It is partially inhibitory for gram positive organisms. Results must be read within 18-24 hours. The eosin Y and Methylene blue dyes in Levine may inhibit some gram positive. These dyes also play a role in differentiating between lactose and non-lactose fermenters due to the presence or absence of dye uptake in the bacterial colonies. Lactose fermenting organisms are visualized as blue-black or brown colonies, whereas lactose non-fermenters appear as colorless, transparent, or amber in color. Some gram positive bacteria such as fecal enterococci and staphylococci will grow on this medium and usually form colorless pinpoint colonies. a. this medium is selective for gram negative bacteria. b. this medium is differential between gram negative lactose and lactose non-fermenting enteric bacteria c. Eosin Y and Methylene blue dyes are the selective component. d. Lactose sugar is the differential component. E. Eosin Y and Methylene blue dyes are also the indicators. |
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| What are the typical colony morphologies for the EMB plates? |
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| E. Coli - blue black with green metallic sheen. The metallic sheen is caused by the large quantity of acid that is produced; the acid precipitates the dyes onto the growth's surface. E. aerogenes: flesh color to pink, centers may be deep brown. Neighboring colonies may run together. S. typhimurium: colorless amber growth S. aureus: pinpoint colorless growth |
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| What are MAC plates used for? |
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| differential and selective. The MAC plates are a differential medium recommended for use in the isolation and differentiation oflactose fermenting organisms from lactose non fermenting gram negative enteric bacteria. Results must be read within 18-24 hours. Colonies of organisms capable of fermenting lactose produce a localized pH drop which, followed by absorption of the neutral red, imparts a dark pink color o the colony, a zone of precipitated bile may also be present due to this localized drop in pH (the medium becomes opaque). Colonies of organisms that do not ferment lactose remain colorless and translucent with a slight browning of the media due to aging of the plate during incubation a. this medium is selective for gram negative bacteria b. this medium is differential between gram negative lactose fermenting and non lactose fermenting enteric bacteria c. crystal violet and bile salts are the selective component. d. lactose sugar is the differential component. e. neutral red is the indicator |
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| What is the typical colony morphology for MAC plates? |
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| E. Coli: a gram negative bacteria and a lactose fermenter, therefore it will produce acid, the indicator neutral red will turn the colony pink. The acid byproducts of lactose fermentation will precipitate the bile salts imparting opacity to the medium. e. aerogenes: a gram negative lactose fermenter the colony will turn pink with no change to the media S. typhimurium: is gram negative. It will grow but does not ferment the lactose, therefore the colony will be colorless. S. Aureus: a gram positive. growth is inhibited. |
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| Account for the microstatic effect produced by low temperatures as compartd to microbicidal effect produced by high temperatures . |
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| Microstatic - low temperatures inhibit enzymes and therefore do not grow microbicidal - high temperatures kill celllar enzymes which become irreversably denatured |
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| cite the advantages of each of the modes of sterilization: tyndallization and autoclaving. |
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| autoclaving - only takes 15 mins and kills all organisms. uses steam which penetrates cell wall more quickly tyndallization - time consuming. can kill off things that die at lower temps while they leave things that grow at higher temps alone. |
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| Discuss the detrimental effects of control agents on the following: the cytoplasm, the cell wall, nucleic acids, and the cell membrane. |
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| They can cause them to be resistant to heat and not be killed by steriliation because they have stregnthened them |
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| Explain why milk is subject to pasteurization rather than sterilization |
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| milk still requires some vegetative cells pasturizing ensures that pathogenic cells are killed without killing all vegetative cells. |
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| Discuss the effects of ionizing radiation on cellular constituent |
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| it can cause them to lose their chemical strctures and forms HO2 free radicals which can combine and form H2O2 which is highly toxic to cells lacking catalase or peroxidase and highly reactive with some cellular constituents resulting in cell damage. |
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| Explain why x rays can be used fro sterilization, whereas ultraviolet rays can be used only for surface disinfection of materials |
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| UV rays have a lower energy content than x rays |
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| Explain the mechanism of action of ultraviolet radiation on cells |
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| It gets absorbed into the cells and causes thymine dimers to form in nucleic acids |
