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MCB 3020
Lab Practical
| Term | Definition |
|---|---|
| simple microscope | one lens |
| compound microscope | two lenses |
| as you close the diaphragm | the light intensity decreases; contrast improves; depth of field increases; limit resolution with oil immersion lens |
| cocci | spherical shaped |
| bacilli | cylindrical shaped |
| helicoidal | spiral shaped |
| diplococci | divide in one plane; pairs |
| streptococci | divide in one plane; chains |
| staphylococci | divide in three planes irregularly; bunch of grapes |
| tetracocci | divide in two planes; square |
| sarcinae | divide in three planes regularly; cube |
| coccobacillus | very short and almost appear spherical, but they are just slightly longer in one direction than the other |
| fusiform bacilli | tapered at both ends, appearing like a football |
| filamentous bacillary forms | grow in long threads |
| cationic dyes | basic, positive charge; methylene blue, crystal violet |
| anionic dyes | acidic, negative charge; acid fuschin, congo red, nirogsin |
| fat soluble dyes | no charge; sudan black |
| insoluble dyes | water insoluble; india ink |
| negative staining | stains background not cell; two dyes used: nigrosin (black anionic dye; repelled by the negatively charged surface of bacteria) india ink (insoluble dye that does not penetrate the cell surface); used for cell size and morphology; NO smear prep |
| simple staining | one dye used to stain all cells the same color; can be used to tell morphology and size (negative staining is better for this); cationic dyes are attracted to the negatively charged surface of bacteria; methylene blue and crystal violet |
| differential staining | causes cells to stain differently based on cell wall properties; gram and acid fast staining |
| gram stain | gram + have a higher peptidoglycan and lower lipid content than gram - cells; stained with crystal violet then fixed with iodine forming a crystal violet-iodine complex; decolorized with ethanol; counterstained with safranin; g+ purple |
| acid fast stain | have a high wax content in their walls, which requires steam to force dye to penetrate cell; steamed in the presence of carbol fuschin and decolorized with acid alcohol; acid fast remain red; non acid fast are colorless then counterst. with methylene blue |
| spore staining | malachite green is steamed into the spores then all other cells are decolorized with water while the free and endospores retain the dye. counterstained with safranin; |
| aerobic organism, produces spores | bacillus |
| anaerobic organism, produce spores | clostridium |
| tube media prep | pour, broth, deep, slant, and fermentation broth |
| natural media | raw materials, chemical composition unknown, NA(melts at 100 and solidifies at 45) |
| synthetic media | chemical composition is known; can be selective or differential |
| selective media | a media which favors the growth of one type of microorganism over another. this is accomplished by either inhibiting unwanted microorganisms or enriching conditions towards preferred microorganisms. |
| differential media | a media which differentiates or distinguishes between different types of microorganisms based on differences in appearance of growth or color changes |
| phenylethyl alcohol agar (PEA; clear) | selects for the growth of gram + microorganisms, + if growth |
| desoxycholate agar (DES; light orange) | selects for gram - microo.; differentiates for lactose fermenters; lactose fermenters produce acid precipitate the bile salts in the media and absorbs the neutral red dye. |
| eosin methylene blue agar (EMB; red) | selects for gram - microo; differentiates lactose +/- microo; lactose + show a color change; can differentiate based on amounts of acid produced; purple and pink bullseye: lactose+; black/blue: mixed+ |
| blood agar (BA; red) | differentiates microo. based on their reactions in blood; gamma hemolysis=no zone of clearing; beta hemolysis=complete zone; alpha hemolysis=partial zone sometimes appears green due to partial reduction of hemoglobin in blood; 5% sheep's RBC |
| starch agar (line or point inoculation; clear) | tests for the presence of exoenzyme, amylase (hydrolyzes starch to simple sugars); Iodine is added to starch plate and appears blue/black when interacting with starch; if amylase +, there will be a colorless area around the colony |
| milk agar (line or point inoculation; cloudy) | tests for the presence of enzyme caseinase (hydrolyzes casein, a milk protein, into amino acid products); breakdown in casein cause the milk plate to lose its white color and become clear around caseinase + |
| lipase agar plate (line or point inoculation; deep blue) | tests for the presence of enzyme lipase (hydrolyzes fat to form glycerol and fatty acids); production of fatty acids lowers the pH just enough to produce a dark blue precipitate when lipase +; indicator is spirit blue |
| sugar fermentation tubes | tests for fermentation of particular sugars; tubes contain the sugar of interest (glucose, lactose, mannitol), pH indicator (phenol red) and Durham tube; |
| sugar fermentation results | if able to ferment the sugar, produces acid which lowers the pH and changes color to yellow from original red color; if makes gas, its trapped in the Durham tube; if alkaline (uses peptone in the broth and not sugar), darkening of the red pH color |
| methyl red (MR) | HCOOH->CO2+H2; tests for mixed acid fermenter; MAF produce drastic amounts of acid from the fermentation of sugars; acid results in the pH dropping below 5.1, so when methyl red is added, it remains red; if -, then yellow; Escherichia |
| voges-proskauer (VP) | HCOOH->acetyl methyl carbinol(acetoin)->2,3 butanediol; tests for 2,3 butanediol fermenter; 2,3BF produce less acid and more neutral products. acetoin is easier to detect than 2,3BF so it tested for with Barrit's Reagents; |
| voges-proskauer (VP) results | when oxygen is present, KOH will react with acetoin to produce a brick red color at the top and indicate that the microo. is a 2,3 butanedoil producer; alpha-naphthol is used to intensify the red color; enterobacter |
| VPI (Barrit's Reagents) | alpha-naphthol |
| VPII (Barrit's Reagents) | KOH |
| catalase | converts 2(H2O2) -> 2(H2O)+O2; can be tested by adding H2O2 to the culture and looking for bubbles, if bubbles, then +; can't be used on blood agar because blood contains catalase |
| oxidase | an enzyme which can oxidize aromatic amines to form colored products; test for oxidase with dimethyl-p-phenylenediamine hydrochloride which when in the presence of oxidase will turn a dark blue/black color on a filter paper |
| motility media (clear) | tests if the bacteria are motile or not; can contain tetrazolium chloride, a growth indicator which turns red in the presence of metabolizing microo, does NOT indicate motility; |
| nitrate broth (clear) | tests for the ability of microo. to reduce nitrate(NO3) to nitrite(NO2); + if red after nitrate 1 and 2; - if red after zinc; + if never red |
| nitrate 1 | sulfanilic acid |
| nitrate 2 | dimethyl-alpha-napthylamine |
| tryptophan broth (indole; clear) | tests for enzyme tryptophanse which converts tryptophane to indole, pyruvic acid and ammonia; indole can be tested with kovac's reagent which will be red ring if + for indole; yellow ring: - |
| Kovac's Reagents | p-dimethylaminobenzaldehyde, amyl or butyl alcohol, and HCl |
| urea broth (yellow/orange) | contains the substrate urea and the pH indicator phenol red; when ammonia is released the pH increases and once above 8.1, the phenol red will appear red;+ appears red; - appears yellow below 6.8; proteus |
| hydrogen sulfide production (H2S) | test for the enzyme cysteine desulfurase which removes the sulfur side chain from cytesine to produce H2S, which when in the presence of iron salts forms a black precipitate; H2S+: black precipitate; proteus: H2S+ |
| SIM | tests for sulfur production, indole, and motility; H2S+ is black precipitate; indole positive: kovac's reagent turns red; motility positive: growth away from inoculation line |
| simmons citrate slant (light inoculation; green slant) | tests for the ability of a microo. to utilize citrate as the sole carbon source; if positive, growth on the media and/or media may turn a deep blue color; indicator is bromo thymol blue |
| phenylalanine slant (PPA; opaque) | tests for the presence of the enzyme phenylanase which converts phenylalanine to PPA and NH3; ferric chloride is added to the media, which in the presence of PPA will appear green |
| litmus milk broth | tests for lactose fermentation, reduction of litmus, presence of caseinase, and the deamination of amino acids to produce NH3; contains the pH indicator Litmus and powdered milk; |
| litmus milk results | acid reaction-pink liquid due to drop in pH from ferm. of lactose; acid curd reaction-pink solid due to acid production and coagulation of proteins; reduction-litmus is reduced to be colorless and the tube appears white; |
| litmus milk results, part 2 | alkaline reaction-blue liquid which is caused when protein breakdown produces amino acids that are deanimated and release ammonia; peptonization/proteolysis-clearing of medium (brown or amber) caused by enzyme caseinase |
| IMViC profile | set of four tests that are used to differentiate between escherichia coli and enterobacter aerogenes; indole, methyl red, voges-proskauer, and citrate. e. coli is + for IM and e. aerogenes is + for ViC |
| gelatin | a protein that solidifies at lower temperatures; stab inoculated and then placed on ice for several minutes; gelatinase +, liquid after being put on ice; gelatinase -, solid after being put on ice |
| Klinger's iron agar slant (KIA; red) | tests for ability to ferment glucose and or lactose, tests for H2S production, and for gas production; contains 0.1% glucose, 1% lactose, iron salts, and phenol red; read results for glucose/H2S in the butt and lactose at the slant; |
| Klinger's iron agar (KIA) results | yellow butt: glucose+; yellow slant: lactose+; black: H2S precipitation; cracking: gas production |
| OF glucose (oxidation; fermentation; green) | tests to determine if a bacteria can use gluc. in an oxidative or fermentative condition; one of the tubes covered in mineral oil to stop air flow; Media contains pH indicator Bromo Thymol Blue which turns yellow if the gluc. is used and acids are made |
| OF glucose results | both tubes yellow: fermentative; both green: oxidative; open green/closed yellow: facultative |
| bismuth sulfide agar (BSA; dull green) | tests for sulfur production; salmonella typhi produces a black or very dark brown, shiny color |
| brilliant green agar (BGA; red) | Differential for lactose/sucrose ferment.; fermenting organisms produce yellow/green colonies and turn the media yellow/green; Non- fermenting organisms produce opaque red/pink/white colonies and turn the media red; indicator is phenol red |
| salmonella/shigella agar (SS; orange/red) | selects for g-; differentiates lactose fermentation; indicator is neutral red; Salmonella usually produces a black colony, Shigella a colorless colony & all lactose positive colonies appear red |
| Desoxycholate Citrate | selects for gram -, differentiates for lactose – microorganisms; indicator is neutral red; some Lactose + colonies do grow but they will appear Red |
| coagulase (tiny amount of clear liquid) | If rabbit plasma becomes clumpy and or solidifies, then bacteria are coagulase+; Test is only valid on gram + staphylococcus aureus like bacteria since gram negative bacteria are able to provide false positive reactions |
| Mannitol Salt Agar (MSA; red) | selects for Staphylococcus due to high salt concentration 7.5%; differentiates for mannitol and staph aureus will turn yellow if organisms are mannitol +; indicator is phenol red |
| Staphylococcus 110 Medium (clear) | contains mannitol and 7.5% NaCl, but lacks Phenol Red as in MSA plate; selects for Staphylococcus and allows for development of natural colony pigment formation unlike in MSA |
| DNase medium (line or point inoculation; light blue) | Tests for exoenzyme DNase which is able to hydrolyze DNA; Zones of clearing around streaks either before or after addition of 0.1M HCl is a positive result for the presence of DNase; indicator is methyl green |
| M-staphylococcus broth | Enriched media containing 10% NaCl, which selects for Staphylococcus because they prefer the higher salt concentration, which inhibits most other organisms |
| endo agar (pink) | Selects for gram –; Differential for lactose, lactose + = red colonies and surrounding medium; Coliforms produce a golden metallic sheen; enterobacters are pink |
| staphylococcus | cocci clusters; gram +; aureus (mannitol positive, coagulase positive, catalase positive, MSA/SM110 growth, nitrate positive); staph infections, Mursa; |
| streptococcus | cocci chains; gram +; beta hemolytic; catalase -; PEA growth |
| gram + pyogenic cocci study | Staphylococcus: Found in nasal membranes, the hair follicles, the skin, and perineum Most strains are penicillin resistant; Streptococcus: Found in pharynx, on surfaces of the teeth, saliva, shin, colon, rectum, and vagina |
| gram - intestinal pathogens study | major concern for public health since the two main pathogens Salmonella and Shigella have the ability to cause enteric fevers, food poisoning, dysentery, and even typhoid fever. |
| standard plate count | accomplished by diluting the sample and growing the bacteria on NA. Counting the # of colonies that grow on NA determines the # of bacteria in the dilution. To determine the population of the original, take the # in the dilution and times by the DF |
| dilution factor (DF)= | A/(A+B) |
| plated DF= | A/1mL |
| direct microscopic count | accomplished by staining a measured amount of milk that has been spread over a known area (usually 1cm^2) on a slide. The slide is examined under oil immersion and the microo. in a field are counted. Several fields are counted to increase accuracy. |
| number of organisms per mL | (average # of bacteria)(microscopic factor)(1/dilution factor) |
| microscopic factor | (area of the film)/(area of the field) |
| breed slide | wells to let the milk dry in a direct microscopic count; xylene washes away fat globules of milk; methylene blue stains cells |
| stage micrometer | a slide with a tiny ruler on it |
| bacterial examination of water | testing to see if there is e. coli in the water supply; presumptive: 9-12 tubes of lactose broth, MPN; confirmative: EMB or endo; completed: lactose broth and NA slants |
| membrane filter method | a faster method of bacterial examination of water that uses a vacuum to pull water through a filter paper to catch microorganisms such as e. coli that can then be placed on a plate; if color changes, + |
| filter paper disc method | Used to compare antiseptics based on their bacteriostatic properties; Relative effectiveness is measured by the size of the zone of inhibition (measured from edge of plate to edge of inhibition zone); formaldehyde |
| antiseptic | applied to living skin or tissue to prevent infection; iodine |
| disinfectants | applied to surfaces, equipment or other inanimate objects; stronger than antiseptics; lysol |
| kirby-bauer method | Used to compare effectiveness of antiseptics, both antibiotics (made naturally) and drugs (man made) which is done under a standardized system against McFarland's standard |
| bacterial lawn | streak the culture across the entire plate |
| pleomorphic | constantly changing shape; club/bat-shaped; corynebacterium |
| staphylococcus vs. streptococcus | catalase and nitrate |
| salmonella vs. proteus | urease |
| e. coli vs. entero | IMViC |
Created by:
JacobGant
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