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Alex Lab Exam
Lab Exam 2
| Question | Answer |
|---|---|
| Way to determine bacterial numbers | Microscopic counts-slides (Petroff Hauser chamber=grided slide), Most Probable Number (MPN), Standard Plate Count (SPC), Turbidity |
| Characteristics of coliforms | G-, facultative anaerobes, non endospore forming, ferment lactose to produce acid and gas, found in intestine of humans, presence of these in water is potential for disease |
| Enteric bacteria | Reside in GI tract and can have G+ enteric like Enteroccocus faecalis |
| G+ enteric bacteria | Enterococcus faecalis |
| G- enteric bacteria | Escheria coli |
| Example of microscopic counts | Sample can be diluted and cells counted with aid of microcope..Petroff-Hauser chamber, grid pattern |
| How are number of bacteria determined in MPN | # determined by relationship of growth parameter to statistical probability. Limited testing where statistical tables have been set up for a particular growth pattern. |
| Explain SPC | Viable counting Series of dilution blanks and incubated for 24-48 hrs # of bacteria in original samples is determined by multiplying number of colonies by dilution factor |
| Why use CFU's | Cells in a chain (like strep) will give rise to a colony on a plate. Uncertainity of how many actual cells are in a colony use Colony Forming Units (CFU) |
| Indirect methods include? | turbidity test |
| Explain relationship of turbidity and cell growth | Growing cells increase in protein, nucleic acid, and mass due to dividing. Measuring these growth factors shows relationship to amount of bacteria. More turbid=higher optical density=more bacteria |
| Optical density relationship to bacteria growth | Amount of light absorbed is proportional to the number of cells present |
| 378 colonies were counted on a plate that received 1.0 ml of the 1:1,000,000 dilution. How many CFU's in original? | 3.8x10^8 |
| 30 colonies were counted on a plate that received 0.1 ml of the 1:1,000 dilution. How many CFU's in original? | 3.0x10^5 |
| How to get 1:10 dilution? | 1ml bateria and add to 9ml of water blank |
| How to get 1:100 dilution? | 1 ml of 1:10 dilution to 9ml h20 OR 0.1ml of original in a 9.9ml blank 10^-2 |
| How to plate 1:10,000 dilution? | plate 1ml of 1:10,000 dilution OR 0.1ml of 1:1,000 dilution |
| Why is it important to perform a plate count in conjunction with a turbidity procedure? | Necessary to determine if cells continue to divide. |
| What is meant by an intestinal indicator species? | A species of bacterium to test for in water that may indicate precense of coliforms. Easier to test because less fasatidious. |
| Why is E.coli a good indicator species? | 1. occurs in intestines of humans and some warm blooded animals 2. can be easily identified by microbial tests. 3. is not as fastidious as the intestinal pathogens Also not normally found in soil or water comes straight from feces E.aerogense can bei |
| What was the procedure used in testing river water? | Most probable number determination |
| What are the three tests used to determine coliform count? | Presumptive, Confirmed, and Completed |
| Describe the presumptive test | 15 tubes of lactose broth are innoculated with measured amounts of water. If gas is seen the it is PRESUMED that coliforms are present Used to determine most probable number of coliforms |
| Describe Confirmed test | Levine EMB agar or Endo agar are inoculated from positive (gas producing) tubes Both media inhibit gram + bacteria Looking to see if G- bacteria will grow |
| Describe appearance of coliforms on Levine EMB agar | Dark pink growth shiny metallic green for Ecoli |
| Desribe Completed test | gram stain from nutrient agar to see if G- no spore forming rod= coliform innoculate Durham tube of lactose broth and if gas is produced = coliform |
| Diseases caused by Coliforms? | Bacterial- typhoid, cholera, dysentery Protozoan-amoebic dysentery, diardiasis |
| Why use Levine EMB? | Eosin-methylene blue inhibits G+ |
| Can G+ grow on Levine EMB | yes G+ ENTERIC bacteria such as Enterococcus aerogenes |
| Why IMViC test? | Used to differentiate between E.coli and E. aerogenes. using indole, methyl red, and coges-proskauer and citrate test Ecoli I+ M+ V- C- E.aero I- M- V+ C+ |
| psychrophiles | supercooled waters 5C-20C |
| mesophiles | body 20C-50C |
| thermophiles | soils, higher temps 50C-80C |
| hyperthermphiles | archaea, volcanic activity above 80C |
| psychrotrophs | bacteria that can grow at temperatures higher or lower than optimum temp |
| Affects of temperature on metabolic facts | Increase in temp: Enzymes begin to denature,membrane lipids destroyed Decrease in temp: chemical activity slows, and can denature, transport slows due to dluidity |
| Optimum growth for Serratia marcescens | 25* (no color at 38) |
| Which bacteria in lab was a thermophile | Geobacillus stearothermophillus >55* |
| Which bacteria in lab was a mesophile | E.coli |
| 3 diseases from spore forming bacteria | Clostridium-tetanus, botulism Bacillus-anthrax, meningitis, UTI, |
| What bacteria produces red pigment on agar | Serratia marcescens |
| Neutrophiles | bacteria grow at neutral pH |
| Acidophiles | grow at acidic pH (pH 1) most fungi and yeast are acidophiles |
| Alkaliphiles | grow at alkaline pH (10 or above) Bacillus is an alkaliphile |
| How does pH affect bacteria growth | H+ ion concentration affects proteins and other charged moluecles in cell. It can affect acharge on amino acids in proteins and result in denaturaion and loss of enzyme activity. |
| How would a sugar fermenter affect the pH | sugar fermenters yield acids and lowers pH |
| How would urea hydrolysis affect pH | Urea hydrolysis produces ammonia and increases the pH making it more alkaline |
| How is pH utilized in food preservation | fermentation of foods can yield acids lowering pH and preventing growth of other microorganisms |
| osmosis | water diffuses from areas of low concentration to areas of high concentration |
| Hypotonic envrionment | solute concentrations are lower on the outside of the cell (higher on inside). water moved INTO cell (to lower concentration) swOllen |
| Hypertonic environment | solute concentration is greater on the outside of the cell. water diffuses OUT of cell shRink |
| Isotonic environment | solute concentration is teh same inside and outside of cell |
| Plasmolysis | resulting in a loss of water, dehydration of the cytoplasm, cell SHRINKS, membrane pulls away from cell wall, irreversable damage hypertonic environments encourage |
| Halophiles | require high concenrtions of salt archaea |
| Halotolerant | growth in moderate concentrations of salt Staphylococcus aureus |
| Osmophiles | able to grow in environments where sugar concentrations are excessive xeromyces (yeast) |
| Obligate halophile vs facultative halophile | Obligate halophile REQUIRES high salt concentrations facultative halophile are able to grow in high salt concentrations |
| Examples of obligate halophile and facultative halophile | Obligate=Halobacterium Facultative=S.aureus |
| Define Ultraviolet light | nonionizing short wavelengths radiation |
| Best wavelength to kill bacteria | the shorter the better 260 nm is most germidcidal because it is specific wavelength at which DNA maximally absorbs UV |
| Pyrmidine dimers | caused by UV light covalent bonds between two adjacent thymine or cytosine molecules in DNA |
| Why are pyrimidines detrimental | deformed so that DNA polymerase cannot replicate DNA strands past site of dimer formation and no transcription |
| SOS system | repair mechanism enzymatically removes dimers and inserts a new pyrimidine molecule in place can end up inserting wrong base and can result in death |
| Factors affecting UV affect | time exposure, presence of material blocking radiation, endospores are more resistant to UV |
| Why are endospores more resistant to UV | acid-soluble proteins bind to DNA and alter conformation protecting it from photochemical damage unique spre photo-product is generated by UV light and functions in enzymatic repair of damaged DNA during germination of endospores |
| Antibiotics vs antimicrobials | Antibiotics are antimicrobials that are produced by microorganisms that inhibit or kill other microorhanisms. Antimicrobials encompass synthetic antibiotics as well as natural |
| Kirby-Bauer method | is used to determine the sensitivity or resistance of a bacterium to an antimicrobial. |
| What specific medium must be used in testing teh effectiveness of antibiotics | mueller-hilton II agar |
| Antiseptics | inhibit microbial growth or kill microorganisms and are gentle enough to be applied to living tissue do no destroy endospores hand washing treating surface wounds |
| Examples of antiseptics | alochols or betadines |
| Disinfectants | chemical agents applied to inanimate objects to kill microorganisms. harsher than antiseptics kill endospores |
| Disenfectants also known as and examples | sterilants and sporocides ethylene oxide |
| Sanitizers | agents that reduce microbial numbers to a safe level but do not completely eliminate |
| Bacteriostatic | agent only inhibits growth of bacterial cells but does not kill them |
| G- are sensitive to | detergents and alcohols because of LPS layer |
| Normal flora | microorganisms that reside persistently on the body naturally human normal bacteria deeply entrenched in the skin and hair follicles and very difficult to remove |
| 3 types of bacteria considered as normal flora | Diptheroids,staphylococci, yeast and fungi |
| Diptheroids | Gram +, non pathogenic ex. Propionibacterium acnes in hair follicles and breaks down sebum to keep skin from drying out |
| Transient bacteria | endospores and endospore fomers and organisms causing temporary infections. Easily removed. |
| Yeast and fungi on skin | normally nonpathogenic but some can cause opportunistic infections and some are dermatophytes (infect hair, skin and nails) |
| Staphylococci on skin | Staphylococcus epidermidis S. aureus (some people)-opportunistic pathogen |
| What is a protease | enzymes that degrade proteins |
| What test tests for proteases | Gelatin stab |
| What is an exoenzyme | an enzyme that functions outside the cell wall to degrade large macromolecules. Can break down proteins, polysacch into amino acids and transport back into cell for metabolic needs ex proteases, DNase, amylase |
| Respiration | organic molecules are degraded into CO2 and water ATP is generated by energy created from a proton gradeint uses oxidative phosphorylation |
| What is oxidative phosphorylation | shuttling electrons down an electron transport chain involving chytochromes facilitates the movement of protons outside of the cell. terminal electron acceptor is O2 |
| What is fermentation | partial breakdown of organic molecules to alcohols, aldehydes, acids, and gases such as CO2 and H2 organic molecules in metabollic pathways serve as terminal electron acceptors uses substrate level phosphorylation |
| What is substrate level phosphorylation | metabolic intermediates in pathways directly transfer high energy phosphates to ADP |
| What does the mixed acid fermentation differentiate | differentiates G- intestinal bacteria |
| What are the reagents in Voges-Proskauer test | Barrits reagent A and B acetoin reacts with Barrits reagent |
| What is Barrits reagent made of | alpha-naphthol and KOH |
| What does Citrate test test for | bacteria that use citrate as the sole carbon source |
| How does citrate test work | organisms degrading citrate must also use ammonium salts in the process. This produces ammonia which is alkaline. Under alkaline conditions pH indicator turns medium dark blue |
| Oxidase test tests for | presence of cytochrome oxidase differentiates bateria that use o2 as terminal e- acceptor indicates aerobic bacteria |
| Reagent in oxidase test | oxidase reagent NNNN-tetramethyl-p-phenylenediamine dihudrochloride |
| What does catalase test test for | bacteria that produce catalase to break to hydrogen peroxide |
| What does the citrate test differentiate between | bacteria that are anaerobes and produce catalase and those that are strict anaerobes are aerotolderant will not have catalase and can no break down the hydrogen peroxide |
| What does nitrate reduction test for | bacteria that use nitrate respiration reducing nitrate into nitrite with nitrate reductase testing for presence of nitrite and gas |
| What bacteria utilize nitrate reduction | Paracoccus Ecoli (facultative) Pseudomonas and Bacillus (aerobic) |
| Enzyme utilized in starch hydrolysis | amylase |
| reagents in nitrate reduction test | A (sulanilic acid) B dimethyl-alpa-napthylamine) |
| what does amylase degrade stach into | maltose glucose and dextrins |
| What is the reagent in Starch hdrolysis | iodine Iodine reacts with starch macromolecule to turn medium dark, if hydrolyzed there will be a clear halo |
| Casein hydrolysis enzyme | protease |
| What is casein | predominant protein in milk |
| What agar is used in caesin | skim milk agar |
| What bacteria hydolyzes casein | Bacillus subtilis |
| What enzyme is utilized in fat hydrolysis | lipases cleave fatty acids from glycerol |
| How does medium work in fat hyrolysis | the release of fatty acids via lipases lowers the pH of agar and produces a dark blue precipitate sometimes can see fatty oil droplets |
| What are endoenzymes | enzymes that work inside the cell |
| What enzyme is used in urea test | urease |
| what bacteria produces urease | G- enterics ex proteus |
| How does urea test work | urease splits urea into CO2 and ammonia ammonia increases the pH making it more alklaline pH indicator phenol red turns bright pink durign presence |
| What bacteria does phenylalanine deamination test for | G- proteus |
| What does phenylalanine deamination test for | tests for the presence of phenylalanine deaminase |
| What is the reagent in phenylalanine deamination | ferric chloride |
| phenylalaline deaminase produces what | breaks down phenylalanine to produce phenylpyruvic acid and ammonia |
| Enzyme in tryptophan degradation | tryptohanase producing indole, ammonia, and pyruvic acid |
| reagent in tryptophan degradation | kovascs reagent |
| what does tryptophan test test for | indole production |
| Enzyme used in sulfide production | cysteine desulfurase breaks down cysteine into pyruvic acid, hydrogen sulfide and ammonia |
| What medium is used in hydrogen sulfide test | Kligers iron agar |
| What does Kligers iron agar contain | ferrous salts that react with hydrogen sulfide to produce insoluble black precipitate |
| What does SIM test for | S=sulfide I=indole M=motility |
| How is indole tested for in SIM | tryptophan break down produces indole and is tested for by kpvas reagent |
| How is hydrogen sulfide tested for in SIM | if cysteine is degraded into hydrogen sulfide it reacts with ferrous salts and produces black precipitate |
| FTM tests for | Oxygen requirement Only aerobic microserophilic or facultative anaerobe |
| Alkaline reation for litmus milk | turns purple or blue has proteases that degrade the milk proteins producing ammonia causing it to be alkaline |
| acid reaction in litmus milk | turns pink ferments lactose |
| litmus milk reduction | culture becomes white |
| coagulation in litmus milk | curd formation due to precipitation and coagulation of proteins |
| peptonization in litmus milk | medium becomes translucent often turning brown characteristic of proteolytic bacteria that degrades milk proteins |
| What does enzyme rennin do | rennin converts casein to paracasein |
| Why can litmus be used as a oxidative-reduction indicator | certain facultative anaerobes can cause the reduction of litmus dye to a colorless form. the colr change is due to a drop in O2 levels and accompanies production of acids. can also occur when bacteria use the due as an alternative e- acceptor |
| IMViC for E.coli | Indole + methyl red + Voges - Citrate - |
| IMViC for E. Aerogenes | Indole - Methyl red - Voges + Citrate + |
| Nitrogen exists in what forms | Nitrogen gas N2 Ammonia NH3, NH4+ Nitrate NO3- Nitrite NO2- and organic nitrogen |
| Define Nitrogen fixation | Nitrogen (N2) is removed from the atomsphere and fixed to organic forms |
| What enzyme is involved in nitrogen fixation | Nitrogenase |
| What bacteria are involved in nitrogen fixation | free-living aerobic Azotobacter and Azospirillum Anaerobic Clostridium |
| Where does nitrogen fixation occur | root nodules by symbiotic association between Rhizobium and legume plants |
| Define Ammonification | organic nitrogen is decomposed to release ammonia |
| How is ammonia released and utilized in ammonification | Organic matter (and animal wastes) is broken down by microorganisms into NH4+ (ammonium ion) this is then further broken down by other microbes into NO2- (nitrite) and then NO3- (nitrate) which is the main nutrient for plants |
| What organisms are involved in ammonification | Proteolytic bacteria (release NH4+ during protein degrade) Proteus vulgaris uses urease to hydrolyze urea to ammonia |
| What enzyme does Proteus vulgaris use in ammonification | urease to hydrolyze urea into ammonia increase pH |
| Define denitrification | nitrogen returns to the atmosphere organisms that are able to return nitrogen back to the atomosphere as N2 NO3- NO2- N2O N2 (nitrogen gas) |
| What enzyme is necessary for denitrification | nitrate reductase |
| What bacteria species is associated with denitrification | Pseudomonas species |
| What is the function of bacterial exoenzymes | Bacteria can not carry out phagocytosis (cell wall) it excretes exoenzymes outside teh cell wall to break down larger molecules that are transported back in for metabolic needs |
| Difference between fermentation and respiration | respiration uses O2 as terminal electron accepter fermentation uses organic molecules as terminal electorn acceptors |
| Why is catalase useful test to differentiate between streptococcus and staphylococcus | streptococi do not have the enzyme catalse to break down H2O2 (anaerobic) but staphylococci has catalase (aerobic) |
| * Name two tests for reaction that produce alkaline end products | citrate and |
| What types of gases can be produced as a result of sugar fermentation | CO2 H2 and ethanol |
| A nonfermentative test in which gas produced indicates a positive test result | catalase 2H2O2-->2H20 + O2 (gas) |
| Which test is used to differentiate btwn fermentative and oxidative metabolism | O/F glucose test |
| What do Kligers iron and SIM media have in common | production of hydrogen sulfide |
| What media is used to test for 2,3-butanediol fermentation | MR-VP broth |
| What media is used to test for carb fermentation | phenol red lactose Kligers iron agar MR-VP broth |
| What media is used to test for casein hyrolysis | skim milk agar |
| What media is used to test for citrate utilization | simmon's citrate agar |
| What media is used to test for hydrogen sulfide producation | Kligers iron agar SIM medium |
| What media is used to test for mixed-acid fermentation | MR-VP broth |
| What media is used to test for triglyceride hydolysis | spirit blue agar |
| What media is used to test for tryptophan degradation | tryptone broth |
| Name antibiotic producers | mold: Penicillium bacteria: Streptomyces Bacillus Actinomyces |
| Potable water- | water that is safe to drink |
| Diseases associated with non potable water | Bacterial Diseases – Typhoid, Cholera, Dysentery Protozoan Diseases – Amoebic dysentery, Giardiasis Helminths and Viruses also can be found in nonpotable water |
| The resolving power of a microscope is a function of | the magnifying power and numerical aperature of lenses |
| Coarse and fine focus knobs adjust the distance between | the stage and objective lens |
| Parfocal | microscope maintains focus when objective mag is increased |
| Condenser | collects and directs the light from the lamp to the slide does not affect magnifying power |
| Diaphragm | with in the condenser regulates the amount of light that reaches the slide |
| Resolving power of lens | the ability to completely separate two objects |
| Oil immersion | reduces light diffraction and mazimizes the numeriall aperature to improve resolution enhances the resolving power of the microscope |
| Aseptic technique | control microbial levels by frequent cleaning disenfecting |
| Two groups of fungi | molds yeast |
| Molds | fungi that contain filaments called hyphae |
| mycelium | mass of hyphae |
| septa | cross-walls that separate the hyphae into indiviual compartments with an organized nucleus and organelles |
| yeast | lack hyphae multiply by budding or fission |
| pseudohyphae | yeast cells forming chains or buds |
| Pure cultures | single kind of organism |
| Pure colony | assumed to be the identical progeny of original cell |
| What microorganism is known for pleomorphism, metachromatic granules and palisade arrangements | Corynebacterium |
| Metachromatic granules | distinct reddish purple grandules within cells that show up when stained with mehtylene blue these are masses of Volutin, a polymetaphosphate |
| Simple staining involves what | single stain to color bacterial cell |
| What are basic dyes and why are they used | Bacteria are negatively charged and have an attraction to cationic chromophores in the basic dyes ex methylene blue color portion of dye is on + charge colors the bacteria |
| What are acidic dyes and why are they used | Acidic dyes have anionic chromophores and has the color portion on the - charge the bacteria repells the dye and is therefore used to see the background or what surrounds the cell ex nigrosin, india ink |
| Gram staining order | 1. Crystal Violet (primary stain) 2. Wash G+ purple G- pink 3. Grams iodine (mordant) 4. Grams 3 (alcohol) 5. Wash 6. Saranin (counterstain) 7. Wash |
| Purpose of mordant (iodine) in staining | complexes with the crystal violet and forms an insoluble complex in G+ cells. |
| How will acid-fast bacteria appear in a gram stain | should appear G+ |
| Spore staining order | 1. Malachite green (sit covered over water bath) (primary stain) 2. Safranin (counterstain) |
| What species of bacteria do endospores belong to | Bacillus Clostridium |
| Protein coat in endospores | exosporium protective protein coat over bacteria |
| What helps to stain mycobacterium | phenol in dye and heat facilitate penetration |
| Acid fast staining procedure | 1. Heat + Carbolfuchsin (primary stain) 2. acid alcohol (decolorize) 3. methylene blue (counterstain) |
| Diseases caused by Mycobacterium | leprosy tuberucolsis |
| Polar (flagella) | single on either end or both |
| Lophotrichous (flagella) | many on either ends |
| Peritrichous (flagella) | all over surface |
| Amphitrichous (flagella) | many at both ends |
| Brownian motion | movement due to molecular bombardment of cells causing cells to shake or "jiggle" not in a vectorial way |
| Monotrichous (flagella) | single |
| Concentration of agar used in semi solid medium for motility | 0.4% |
| solid agar medium concentration | 1.5% |
| EMB agar selective agents | eoisin methylene blue dye |
| EMB differential agents | lactose dark pink or green sheen if ferments can't ferment light pink or white in color |
| What kind of organisms can grow on EMB (selects for) | G- or G+ enteric bacteria |
| MacConkey agar selective agents | bile salts selects for G- and enteric G+ |
| MacConkey dfferential agent | lactose red colonies if fernemnt lactose can't ferment light or white |
| MSA selects for what organism | Staphylococci because it is halotolerant |
| Selective and differential agents in MSA | mannitol salt has high contentrations of salt only some organisms can grow on If able to ferment mannitol will have yellow zone |
| Autoclave | 121 degrees celcius 15 minutes 15 psi |
| Examples of standard plate counts (SPC) | dilution series |
| Obligate aerobes | Must grow in oxygen Pseudomonas and Bacillus |
| Microaerophiles | lower concentration of oxygen (2-10% rather than 20%) Helicobacter pylori |
| Facultative anaerobe | grow well aerobically but have capacity to grow anaerobically is O2 not present can switch to fermentation E.coli |
| Aerotolerant | tolerant of O2 but do not require strictly by fermentation not by respiratory lack catalase E.faecalis Strep. pyogenese |
| Obligate Anaerobes | absoltely no oxygen no catalase or superoxide dismutase Clostridium |
| Media used to test oxygen requirements | Tryptone glucose yeast exact (TGYA) Fluid thioglycollate |
| MIC | minimal inhibitory concentration lowest concentration of drug to prevent pathogen growth |
| MLC | minimal lethal concentration lowest concentration of drug that would kill pathogen |
| Microbiostatic | reversibly inhibts growth MIC:MLC is less than or equal to .25 |
| Microbiocidal | irreversibly kills pathogen MIC:MLC is greater than or equal to .25 |
| Why is MIC/MLC necessary | prior to chemotherapy it is important to determine the relative resistance of pathogen. Helps to effectively inhibit pathogen and minimize drug toxicity |
| Process of denitrification | anaerobic bacteria utilize Nitrate as e- acceptor by way of enzyme nitrate reductase reducing nitrate to nitrite Some bacteria can further reduce nitrite to N20 and then N2 releasing dinitrogen gas N2 |
| Concentration of salt for growth of Halophiles vs Halotolerant (facultative halophile) | Halophiles require 15-20% NaCl Facultative halophiles are able to grow in moderate [ ] of 11%NaCl |
| Asexual spores of fungi | mitotic division of single parental cell -Sporangiospores (motile and nonmotile) -Conidia (nonmotile) |
| Sexual spores of fungi | fusion of 2 parental nuclei followed by meiosis -Zygospores -Ascospores -basidiospores |
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