Microbiology Lab Test 2

define reduction, oxidation

OIL RIG (oxidation is loss, reduction is gain)

Fluid Thioglycollate medium

- well adapted for cultivation of strict anaerobes and miroaerophiles -sodium thioglycollate and L-cystine reduce oxygen to water, resazurin is used as an indicator of oxygen

Identify the composition of the decolorizer. Explain why acid-fast bacteria are not decolorized.

-3% HCl in 95% ethanol -the mycolic acid gives acid-fast cells resistance to decolorization

List five groupings of microorganisms according to their oxygen tolerance. Know the characteristics of each group.

1. Obligate aerobes 2. Facultative anaerobes 3. Obligate anaerobes 4. Aerotolerant anaerobes 5. Microaerophiles

Evaluate differences between the Kinyoun and Ziehl-Neelsen methods of acid-fast staining.

K: does not use steam to stain ZN: uses steam to stain

Describe the difference between Kligler Iron Agar and Triple Sugar Iron Agar.

KIA contains 2 carbohydrates -glucose -lactose TSI contains 3 carbohydrates -glucose -lactose -sucrose

List the primary types of products which bacteria may produce from fermentation.

Lactic acid Ethanol Butyric acid Propioic acid 2,3-Butanediol Mixed acids

Understand what the reversion of a reaction is and how it might affect the results of both the Phenol Red Broths and KIA.

reversion: as the glucose diminishes, the organisms located in the aerobic region (slant) will break down amino acids, producing NH3 and raising the pH. -a high pH will change the color to make it appear that it did not ferment -fermentation brings down the pH and changes the color, which may give a false reading

Identify the location of spores within the sporangium. Be able to identify terminal, subterminal, and central spores.

terminal: at the end of the cell subterminal: between the middle and end of the cell central: in the middle of the cell

List the reagents utilized in the Kinyoun and Ziehl-Neelson procedures and identify the purpose of each reagent.

K: -primary stain- carbolfuchsin -decolorizer- acid alcohol -counterstain- brilliant green ZN: -primary stain- carbolfuchsin -decolorizer- acid alcohol -counterstain- methylene blue

Prepare mixtures of acid-fast and non-acid fast bacteria, stain them with the Kinyoun or Ziehl-Neelson procedure, and learn to interpret the results accurately

K: acid fast are reddish purple, non acid fast are green ZN: acid fast are reddish purple, non acid fast are blue

Aerobic respiration

a form of respiration that requires oxygen as an electron acceptor in order to generate ATP

Identify the group of organisms which can be differentiated by the IMViC tests.

coliform

Understand how Kligler Iron Agar can be used to differentiate groups of species.

-based on organisms ability to ferment the lactose present in the medium -for Gram-negative enteric organisms that ferment in glucose

Understand why both forms of fermentation may be tested using the same inoculated and incubated broth tube.

-both are fermenting the nutrient medium containing 0.5% glucose -they way they ferment determines the difference in the tests MR and VP

Know the purpose of catalase and superoxide dismutase and how these relate to aerotolerance.

-catalase turns hydrogen peroxide into water and oxygen -superoxide dismutase converts superoxide radicals to hydrogen peroxide -these enzymes allow an organism to live in the presence of oxygen

Candle jars

-divide plate into 4 sections -inoculate one line of bacteria per section -place plate in jar -light candle and wait for it to go out

Explain the role mycolic acids theoretically play in the acid-fast staining reaction.

-gives the cells a higher affinity for the primary stain -gives the cells resistance to decolorization by an acid alcohol solution

Facultative anaerobes

-grow in the presence or absence of oxygen -when oxygen is available, respire aerobically -when oxygen is not available, respire anaerobically or ferment -prefer to grow at the top, but can be found throughout -(EC,SM)

enterics

-gut flora -rod-shaped gram-negative bacteria of the family Enterobacteriaceae -occur normally or pathogenically in intestines of humans and other animals

Define and describe photoreactivation.

-light repair -the repair enzyme, DNA photolyase, is activated by visible light and monomerizes the dimer by reversing the original reaction

Understand why it is important to time how long it takes for the candle in a candle jar to go out.

-measuring how long it takes for the oxygen to be removed from the jar

Accurately appraise the redox potential of a GasPak anaerobic system. Identify the redox potential indicators.

-method used in production of an anaerobic environment -used to culture bacteria which die or fail to grow in the presence of oxygen -indicator is a button that turns either white (oxygen present) or blue (no oxygen present)

Understand why it is important to have nutrients other than the fermentable carbohydrate in the Phenol Red Carbohydrate Broth.

-not all bacteria can utilize the fermentable carbohydrates -the ability or inability of a particular species to ferment a particular carbohydrate depends on the presence of enzymes needed for a particular fermentation pathway

Aerotolerant anaerobes

-organisms that do not require oxygen and are not aversely affected by it -can be found uniformly throughout the medium -(Efl)

Obligate aerobes

-organisms that require oxygen to survive -grow at the top of medium where oxygen is most plentiful -(ML,CV)

Indicate the function of cytochrome c oxidase in a bacterial cell.

-oxidizes cytochrome c -catalyzes the reduction of cytochrome c

Obligate anaerobes

-oxygen is toxic -can be found at the bottom of medium -(Clos)

Understand why water (moisture) accumulates in the candle jar after the lid has been tightened down

-palladium catalyzes a reaction between the hydrogen and free oxygen in the jar to produce water. -removal of free oxygen creates anaerobic conditions

Understand the purpose of an uninoculated control when running metabolic studies.

-shows the medium in an aerobic environment when uninoculated -allows you to clearly see the fermentation in an anaerobic environment in contrast -helps to ensure accuracy of interpretation.

UV-A

-the longest wavelengths -range from 400 to 315 nm

Understand why the amount of glucose is limited as compared to lactose (and sucrose) in KIA and TSI.

-the lower concentration of glucose allows for detection of utilization of this substrate alone -glucose is a monosaccharide, it will be used first

Understand why some bacteria are less susceptible to UV irradiation than others. (For example: Understand why UV light is less successful at killing Bacillus spp.(BSN) than Serratia macescens(SM).)

-the presence of endospores, which are less susceptible to UV irradiation, makes BSN more resistent than SM

Stab cultures

-use a needle and agar deep stabs -promotes growth of a broad range of organisms -tubes are boiled to remove oxygen, needle used to allow as little air as possible into the agar

Understand why a section of each experimental plate was not exposed to UV irradiation.

-used as a control to compare the other sections to

Explain the function of zinc in the test for nitrate reduction.

-used to determine if the nitrates were reduced past nitrite stage -red color with addition of zinc: nitrates were not reduced -no color after zinc: nitrates were reduced beyond nitrites to ammonia or nitrogen gas

UV-B

-wavelengths between 315 and 280 nm

UV-C

-wavelengths range from 280 to 100 nm -most detrimental to bacteria -exposure for more than a few minutes results in irreparable DNA damage and organism's death

Know and understand the purpose of each step in the Schaeffer-Fulton endospore staining procedure.

1. The cells are stained with Malachite green using steam to force the stain into the spore 2. Decolorized with water to remove stain from cells but not spores 3. Safranin is used to counterstain

Prepare a mixture of spore forming bacteria and non-spore forming cocci, stain these slides using the Schaeffer-Fulton procedure and learning to interpret the results accurately.

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Understand the reason for using a drop of serum to prepare the smear for the acid fast stain.

helps the "slippery" acid-fast cells adhere to the slide

Understand why anaerobes are inhibited/killed by oxygen.

lack of catalase and superoxide dismutase

Identify three characteristics of spore formation used by microbiologists to help identify bacterial spore formers.

location in cell shape size relative to cell

Understand the meaning of the term "fastidious" as it relates to bacterial culturing

organisms that will only grow when certain requirements are meet such as nutrients, temperature, pH, salinity, oxygen

spore mother cell

responsible for producing and housing the endospore

Understand why all aerobic bacteria are not oxidase positve

some have a different terminal oxidase system that is not aerobic respiration

Understand why the presence of a bubble in the durham tube of a nitrate broth is not definitive for nitrate reduction when working with an organism capable of fermentation.

the gas bubble could be a product of fermentation, which would make the source is unknown

Understand the purpose of a durham tube in Phenol Red Broths.

to indicate gas production

Understand the purpose of a durham tube in the nitrate test.

to indicate gas production

Understand why it is necessary to repeatedly vortex the VP test over 30 minutes.

to oxygenate it

Understand why tubed media are boiled for at least 10 minutes prior to inoculation or should be used soon after initial preparation. (agar shake cultures, stab cultures, Fluid Thioglycollate medium)

to remove air and oxygen from the medium

Identify the sources of error that might produce false positives in the oxidase test.

waiting too long to read the test

sporagium

where spores are produced

Understand the relationship between the enzyme nitrate reductase and anaerobic respiration.

-anaerobic respiration is the reduction of an inorganic molecule -nitrogen is that inorganic molecule -thus nitrate reductase is what reduces NO3 to NO2

Identify genera that include acid-fast species

Mycobacterium

Enterobacteriaceae

-a large family of Gram-negative bacteria that includes Salmonella, Escherichia coli, Yersinia pestis, Klebsiella and Shigella -Many live in the gut -facultative anaerobes, fermenting sugars to produce lactic acid and various other end products -reduce nitrate to nitrite -lack cytochrome c oxidase

Oxidation of carbohydrates

the metabolic process by which an organic molecule acts as an electron donor and one or more of its organic products act as the final electron acceptor in respiration

keratin

the protein that makes up the tough outer covering of the spore

What two metabolic pathways lead to the production of hydrogen sulfide?

the reduction of thiosulfate in the medium and breakdown of cysteine

Understand why acid-fast bacteria are considered Gram nonreactive.

the waxy wall of acid-fast cells repels typical aqueous stains

Understand why aerotolerants would not use catalase.

they do not use oxygen, so they do not need to convert hydrogen peroxide into water and oxygen via catalase

Understand why some microbiologists advocate using 1% carbohydrate rather than 0.5% carbohydrate in phenol red broths.

to ensure against reversion of the reaction due to depletion of the carbohydrate

Interpret the results of the experiment: How does UV irradiation affect CV,SM, BSN? How does the ability to complete photoreactivation change those effects?

-BSN was least affected because of sporulation -SM grew better than CV with just UV light because SM has better dark repair -the exposure to visible light improved the survival of CV but not SM because CV uses light repair

Differentiate catalase and peroxidase.

-Catalase converts hydrogen peroxide into oxygen and water -Peroxidases are the plant enzymes that use peroxide to break down bacteria and other harmful material

Accurately appraise the redox potential of tubes of Fluid Thioglycollate Medium. Identify the redox potential indicators.

-Sodium thioglycolate in the medium consumes oxygen and permits the growth of obligate anaerobes -indicator is resaruzin, which turns pink when oxidized and colorless when reduced

Differentiate spontaneous and induced mutation.

-Spontaneous mutations occur infrequently and randomly in natural environment -induced mutations occur due to exposure to mutagens (both chemical and physical)

Anaerobic respiration

-a form of respiration using electron acceptors other than oxygen -still uses the electron transport chain

Fermentation

-a metabolic process that converts sugar to acids, gases and/or alcohol -occurs in yeast and bacteria but also in oxygen-starved muscle cells

Microaerophiles

-high oxygen level is toxic but need a little oxygen -can be found somewhere near the middle or upper middle region of the medium

Shake cultures

-inoculate with loop then gently roll to spread -allows bacteria to spread throughout medium -heated or boiled to drive oxygen out

Define and describe excision repair (dark repair) of DNA.

-involves a number of enzymes -the thymine dimer distorts the sugar phosphate backbone -this is detected by UvrABC endonuclease that breaks two bonds -helicase removes the 14-nucleotide fragment, leaving single stranded DNA -DNA polymerase I inserts the appropriate complementary nucleotides -DNA ligase closes the gap between the last nucleotide of the new segment and the first nucleotide of the old DNA

Understand why UV irradiation has limited application for sterilizing materials.

-it doesn't penetrate materials such as glass and plastic well -bacterial cells have mechanisms to repair UV damage -surface has to be flat

GasPaks

-place plates in bag -open GasPak -seal immediately -creates anaerobic, carbon dioxide rich environment -white button= no oxygen is present

Understand the nitrate/nitrite test and be able to accurately interpret the results.

-reduction of nitrate is generally an anaerobic respiration in which an organism derives its oxygen from nitrate -in this test, if the organism reduces the 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 -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 were reduced beyond nitrites to ammonia or nitrogen gas

Understand why adding a small amount of agar to the Thioglycollate Medium would be beneficial. (Small amount would be enough to slightly thicken the medium but not solidify it.)

-to slow oxygen diffusion into medium -air moves slower in solids than oxygen

Understand how UV light harms bacteria.

-when DNA absorbs UV radiation at 254 nm, it is most lethal to bacteria -energy creates new covalent bonds between adjacent pyrimidines: C-C, C-T, T-T -pyrimidines dimers: distort the DNA molecule and interfere with DNA replication and transcription

Prepare smears of various spore-forming bacteria and stain these using the Schaeffer-Fulton procedure and learn to interpret the results accurately.

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oxidase

Any enzyme that catalyzes an oxidation-reduction reaction involving molecular oxygen as the electron acceptor

Identify the two bacterial genera that form endospores.

Bacillus and Clostridium

Describe how the toxins hydrogen peroxide and superoxide radicals may be formed and how bacterial cells convert them to non toxic substances.

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.

Prepare and evaluate cultures of each of the test media.

Phenol Red Broth: -inocculate tubes: yellow=acid (fermentation) red= no fermentation (neutral) pink=alkaline (deaminating protein, producing ammonia) MR-VP: inncoulate saline with pure culture and incubate for 24 hours, separate into 2 tubes and add Methyl red to one (Red=mixed acid fermentatation) and Reagents A and B to the second (Red= 2,3 butanediol fermetation) KIA: stab butt with needle then zig-zag on slant yellow=fermentation red=catabolized peptone or proteins and alkaline products black precipitate=sulfur reduction

Differentiate between the lack of sensitivity and the lack of specificity and how each might affect the interpretation of results of metabolic tests.

Sensitivity: an inability to detect small amounts of the chemical or organism in question would yield a false negative result and would be a result of inadequate sensitivity of the test. Specificity: An inability to discriminate between the chemical or organism in question and similar chemicals or organisms would yield a false positive result and would be the result of inadequate specificity of the test. The closer the sensitivity and specificity is to one, the more useful the test is.

Identify the one known spore forming coccal genera.

Sporosarcina

Understand how each of the methods used create environments that are anaerobic and/or have low oxygen tension/increased carbon dioxide concentration.

Stab cultures heated or boiled drive oxygen out. Old tubes will need to be reheated to remove oxygen that may have gotten into it. Water, phyglycolaid will reduce any oxygen Resaruzin- indicator will turn pink to indicate how much air has defused Gas pack- white indicator button = no oxygen, blue= contaminated. Methylene blue turns blue in presence of oxygen. releases carbondioxide capanophiles enviroment, sorbic acid, vitamen C removes the oxygen.

Name at least one bacterial genus that tolerates oxygen but does not produce catalase.

Streptococcus

Coliform

a gram negative, aerobic or facultative anaerobic rod which produces gas from lactose within 48 hours.

cyptobiotic

a state in which there is no metabolic activity

Understand why a Phenol Red Broth base medium would be inoculated in addition to the PRB tubes containing carbohydrates when examining the fermentative abilities of an organism.

as a control, to see if the inoculum changes the base medium

Understand the characteristics of capnophiles as they relate to aerotolerance.

microaerophiles that can survive only if carbon dioxide levels are elevated

Understand the difference between mixed acid fermentation and butanediol fermentation. Know which portion of the MRVP reaction tests for each type of fermentation.

mixed acid fermentation: anaerobic fermentation where the products are a complex mixture of acids ????