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Micro Lab Exam 1
| Question | Answer |
|---|---|
| Basic dyes | Have positive charge and can penetrate the bacteria because of the attraction between charges (Ex: Methylene blue) |
| Acidic dyes | Have negative charge and cannot penetrate bacteria because of repulsion between charges. Stains the background (Ex: Eosin, Nigrosin) |
| Negative staining | Will stain the background of the slide but not the organism (Nigrosine/India Ink used for negative stain exp.) No heat-fixaiton. Useful for determining cell morphology and size |
| Capsular staining is a combination of two staining techniques: | *Negative stain: Congo red to color background *Simple stain: Maneval's stain to color the cell proper of the bacteria |
| Capsules.. | Do not take up stain and appear colorless between the dark background and the stained bacteria. They increase virulence in some organisms, prevent dehydration due to attraction of water, and attachment to host cells. |
| The capsule polysaccharide layer is generally referred to as a _________ | glycocalyx |
| The endospore stain is | A differential stain used to detect the presence and location of spores in bacterial cells |
| Which two bacterial genus produce endospores? | Bacillus and Clostridium |
| All endospore formers are typically gram _____ | positive |
| The exosporium of endospores is made from the protein ________, which resists staining | Keratin |
| What is the primary stain for endospore staining? Counterstain? | Malachite green; Safranin |
| Acid-fast stain is | A differential stain used to detect presence of mycolic acid |
| Mycobacterium are | Rod-shaped, gram positive bacteria which grow around 30*C |
| organisms of the genus mycobacteria have a unique waxy lipid layer called ___________ | mycolic acid |
| Mycolic acid binds to the __________, creating a waxy, hydrophobic layer resistant to dehydration | Peptidoglycan of the cell wall |
| What is the primary stain for acid-fast? | Carbol Fuchsin and phenol mix. Phenol helps stain penetrate waxy layer of the lipid. Carbolfuchsin is lipid soluble so it can enter waxy layer. |
| ________ is used as a decolorizer of non acid-fast cells | Acid-alcohol |
| Mycobacterium are considered to be _____ | acid-fast since it is difficult to decolorize them |
| __________ is used as a counterstain in order to stain the non acid-fast bacteria | Methylene blue |
| Acid-fast bacteria appear __________; Non acid-fast bacteria appear __________ | Red; Blue |
| _________ is used to classify bacteria as gram positive or gram negative | gram stain |
| Gram-positive bacteria appear _________; Gram-negative bacteria appear _________ | Purple; Red |
| Peptidoglycan is made from the sugar derivatives: | N-AM and N-AG bonded by beta 1,4-glysodic bonds |
| Gram positive bacteria have _______ in their cell walls | Teichoic acids |
| Gram negative bacteria have _______ in their outer membrane | Lipopolysaccharide layer (LPS aka endotoxin) |
| What is the primary stain for gram stain? | Crystal violet |
| In gram stain, _______ forms an insoluble compound with crystal violet | Iodine. |
| The peptidoglycan layer of gram-positive bacteria.... | Prevents decolorization because the alcohol causes dehydration and shrinks the cell wall, so the pores in the wall close and the dye becomes trapped. |
| In the peptidoglycan layer of gram-negative bacteria... | Alcohol can move through the lipid bilayer so dehydration does not occur, and the dye can easily be washed away |
| What is the mordant for gram staining? | Iodine - Forms an insoluble compound when interacted with crystal violet |
| What is the decolorizing agent in gram stain? | 95% ethanol - washes dye in gram-negative, but not gram-positive |
| What is the counterstain for gram staining? | Safranin - Stains all the bacteria (gram-negative appear red under microscope) |
| Which stain is used for flagella staining? | Leifson's stain |
| What does leifson's stain contain? | Pararosaniline (stain agent) and tannic acid (mordant) |
| Describe brownian motion | Organism vibrates due to molecules hitting the cell. Organism will remain in one place |
| describe true motility | Direction, independent movement over greater distances |
| Flagella rotate _________; Cell rotates _______. Upon change of direction, the cell will ________ | Counterclockwise; Clockwise; Tumble |
| What are the three methods of motility determination? | *wet Mount *Hanging drop *Motility test media (semisolid media) |
| What does the motility test medium contain? | TTC (triphenyl tetrazolium chloride) |
| Describe TTC | Colorless and soluble when oxidized; Red when reduced |
| What are the three consistencies of media? | *Liquid *Solid (1.5% agar concentration) *Semisolid (0.4-0.5% agar concentration) |
| What are characteristics of a good solidifying agent? | 1) Not utilized by microorganisms 2) Doesn't inhibit bacterial growth 3) Doesn't liquefy at room temperature |
| What are the major macronutrients of organisms? | C, N, P, S, K, Mg, Na, Ca |
| What are the major micronutrients of organisms? | Metals (such as iron specifically) |
| Autotrophs | Can utilize carbon in CO2 to synthesize all materials and utilize light as source of energy through pigments |
| Heterotrophs | Mush have one or more organic compounds for carbon and utilize chemicals for source of energy |
| Minerals... | require metals in small amounts; Minerals are used in compounds and as co-factors in reactions |
| Growth factors... | Are essential compounds that organisms cannot syntehsize (ex: amino acids and vitamins) |
| Pathogens like an ________pH of _____ | Alkaline; 7.3 |
| Many organisms grow best at pH ____ or a little less | 7.0 |
| Describe synthetic/defined media | Exact composition is known; has been synthesized; chemical compounds used are highly purified and defined |
| Describe non-synthetic/complex media | Exact composition is not known; Utilizes compounds that include all the nutrients but they are not defined |
| Describe selective media | Media that allows only certain organisms to grow. Selection is based on absence of certain nutrients which prevent growth of some but not all bacteria and presence of toxic or inhibitory substance (like salt, crystal violet, methylene blue & antibiotic) |
| Describe differential media | Media that contain substances which change the appearance of one species allowing for differentiation (Ex: EMB [Both selective and differential]) |
| Describe minimal media | Media that contains the basic requirements but has an added amino acid such as salts, a carbon source and a nitrogen source |
| Describe enriched media | Media that contains all the basic requirements but has added acids, vitamins and minerals from sources such as peptone or yeast extract |
| What are the different methods of sterilization? | *Autoclaving - Steam sterilization (high temp and pressure) *UV irradiation - Utilizes ultraviolet light *Ethylene Oxide Gas - Generally used for plastic surgical supplies that cannot be autoclaved |
| Autoclaving media for too long of a time _____ | tends to cause glucose and other sugars to degrade |
| What is a pure culture? | Culture containing only one kind of microorganism (always use aseptic technique) |
| What are the different methods for obtaining pure cultures? | *Streak plate method *Pour plate method |
| What are the different types of streak plate methods? | *Quadrant streak - streaks on 4 corners of agar *Radiant streak - radial streak around agar *T Streak *Continuous streak - Streak all around agar |
| Agar plates are incubated ________ | Agar-side up to prevent condensation which may cause organism to run together and defeat purpose of isolation |
| Describe Phenyl Ethyl Alcohol agar (PEA) | Selective media; Inhibits growth of G- Bacteria; Selects for G+ |
| Describe Mannitol Salt agar (MSA) | Selective and differential media; contains mannitol,7.5%NaCl, and phenol (ph indicator); |
| Some species of staph (pathogenic) have the ability to ferment ________ | Mannitol. Acid is produced if mannitol fermentation occurs |
| Describe the color changes of phenol red when detecting pH | Yellow = 6.8 or below Red = 7.4-8.4 Pink = Above 8.4 |
| If an organism is ___________, then it will grow well on MSA but won't ferment mannitol | Non-pathogenic |
| If an organism does not grow well in MSA, then it is probably ________ | Not staph (pathogenic) |
| Describe MacConkey agar | Selective and differential; Contains lactose, bile salts, neutral red and crystal violet; Inhibits G+ (due to bile and crystal) |
| Describe neutral red in MacConkey agar | Red = pH below 6.8 (indicative of lactose fermenters) Colorless = pH above 6.8 (indicative of non-lactose fermenters) |
| Describe Eosin methylene blue agar (EMB) | Selective and differential; Contains peptone, lactose, sucrose, eosin, methylene blue; Sugars provide fermentable substrubes that promote growth of fecal coliforms; Methylene blue inhibits G+; |
| Eosin is ________ | An acidic dye that differentiates on the basis of fermentation |
| Describe color indications of eosin | *Green metallic sheen-deep purple = Fecal coliform *Pink = Low acid production/slow fermenters *Colorless/same color as media = nonfermenters |
| Describe Hektoen Enteric agar (HE) | Selective and differential; Contains peptone, lactose, sucrose, salicin, bile salts, sodium thiosulfate, ferric ammonium nitrate, bromthymol blue, and acid fuchsin; Sugars provide fermentable substrates that promote growth of fecal coliforms; inhibits G+ |
| What inhibits growth of G+ bacteria in HE agar? | Bile Salts |
| What is the purpose of bromthymol blue and acid fuchsin in HE agar? | Dyes that act as color indicators to indicate the fermentation of sugars and production of acids that lower the pH of the medium |
| Describe different color indications of HE agar | *Yellow - salmon-pink = enterics that produce acid from fermentation *Blue-green colonies = Bacteria that do not ferment any sugars *Black precipitate = Bacteria that reduce sulfur to H2S to form colonies |
| What is the purpose of sodium thiosulfate in HE agar? | Acts as source of sulfur |
| What is the purpose of ferric ammonium citrate in HE agar? | Reacts with H2S produces by the reduction of sulfur to form black precipitate |
| The molds aspergillus niger and penicillium notatum belong to which group? | Deuteromycetes |
| The mold rhizopus stolonifer belongs to which group? | Zygomycetes |
| What kind of hyphae do aspergillus niger and penicillium notatum have? | Septate |
| What kind of hyphae does rhizopus stolonifer have? | Conenocytic and nonseptate |
| What kind of spores do aspergillus niger and penicillium notatum contain? | None |
| What kind of spores does rhizopus stolonifer have? | Zygospores |
| What kind of asexual spores does aspergillus niger and penicillium notatum and rhizopus stolonifer have? | Conidia |
| What are the classifications of fungi? | Yeast and molds |
| Mycology is the study of? | Fungi |
| Describe some characteristics of fungi | eukaryotic, non-motile, non-photosynthetic, cells walls of chitin, reproduce through spores, lack tissue differentiation |
| What are saprophytic fungi? | Fungi that decompose dead organic matter |
| What are heterotrophic fungi? | Fungi that produce exoenzymes that digest nutrients in the environment then absorbs the products |
| What is mycelium in molds? | Mass of intermeshed hyphae (seen macroscopically) |
| What is hyphae in molds? | Microscopic filaments that may (septate) or may not (nonseptate) have walls |
| Yeasts have _________, which are multicellular structures produced through budding | Pseudohyphae |
| Describe the characteristics of spores in yeasts | Produced by budding which is an outpouching of a cell, smaller in size than parent cell, may or may not stay attached to parent cell |
| Which media is used to grow molds? Describe the media. | Sabouraud's Dextrose Agar (SDA). Has a pH of 5.6 which inhibits bacterial growth |
| The slime-mold culture method is used to grow molds because | wet mounts and simple transfer of hyphae does not provide the structure necessary for identification due to distortion or destruction of these structures during transfer. This allows conidiophores to grow on single horizontal plane |
| What kind of stain is used for preparing slides of slime-mold culture? | Lactophenol cotton blue |
| Describe direct measurement for counting the number of organisms in a culture | Dilute the organisms and count them on microscopic fields on a slide |
| What are the two types of indirect measurement for counting the number of organisms in a culture? | Quantitative plating (viable organisms) and Turbidity measurement (Living and dead cells) |
| How do you calculate dilution factor (DF)? | Volume added to dilution blank / total volume in tube |
| What is final dilution factor (FDF)? | The product of individual dilutions |
| How do you calculate original cell concentration (OCC)? | # of colonies / (Volume plated X FDF) |
| What does CFU stand for? | Colony forming units (# of viable cells that can make a colony) |
| What measurement is obtained for the spectrophotometer? | % transmittance |
| As % transmittance increases, concentration and absorbance _________ | decreases (inversely proportional) |
| How do you calculate absorbance? | 2 - log(%transmittance) |
| Absorbance is also called ________? | Optical density (OD) |
| Absorbance is directly proportional to? | The concentration of the sample |
| Why is it important to quantify bacteria? | For some experiments, it is necessary to know exact # of bacteria present in culture. For every bacteria, a certain concentration is necessary to cause disease. Some bacteria have to be present in greater numbers than others |
| Which culture was used for the dilution method of standard viable counting? | E. Coli |
| What dilution blank was used in the standard viable count lab? | Saline blanks (0.85% NaCl, 9.9 ml) |
| What types of pipettes are used in a microbiology lab? | Serological pipettes and Mohr pipettes |
| What are the types of serological pipettes? | TC (to contain [expel all volume down to tip then last drop must be blown out using pump]) and TD (to deliver [expell all volume down to tip but last drop NOT blown out]) |
| Describe serological pipettes | have graduations down to the tip; temperature-calibrated (volume markings on side of pipette are accurate within certain temp. range [usually 20-25*C (68-77F)]) |
| Describe Mohr pipettes | Not graduated down to the tip so fluid must be only expelled to the last calibration line |
| The plates that were counted in the standard viable count lab had to be statistically significant. How many colonies is statistically significant? | 30-300 |
| Describe viruses | small, non-celluar, intracelluar parasites. Not grown on normal media because they require a host to replicate their DNA/RNA; Can infect both prokaryotes and eukaryotes |
| What are phages? | Viruses that infect bacteria (AKA bacteriophages) |
| Phages that parasitize E. Coli are referred to as _______? | Coliphages |
| What are lytic viruses? | Virulent viruses that enter and take over cell, replicate then lyse the cell for release |
| What are lysogenic viruses? | Temperate viruses that enter the cell, integrate into the host chromosome. Cell replicates normally then eventually viruses replicate and lyse the cell to be released |
| Viruses are observed with what type of microscope? | Electron microscope |
| Normally, viruses are quantified by their effect on the host cells, so you would count the ________? | Virus infection units (VIU's) |
| ______ is the smallest unit cause an effect with a host | virus infection units |
| __________ is utilized to grow isolated plaques of phage particles within a lawn of bacteria | Plaque assay |
| What is plaque in the plaque assay? | The zone of lysis/growth inhibition in a lawn of bacteria. They are clear areas in the zone |
| 1 Plaque is how many viruses? | 1 virus |
| 1 Virus is how many plaques? | 1 Plaque |
| What does PFU stand for? | Plaque forming units |
| Why is the efficieny of count from the assay always less than with an electron microscope? | Because efficiency of infection by viruses is not always 100% |
| What are the three steps used to isolate bacteriophage from sewage (in order)? | Enrichment(increases # of phage in sewage), Filtration(separates E.coli from phage), Seeding(plating the dilution of the filtrate with E. coli to see evidence of phage and to find concentration of phage in original filtrate) |
| What kind of medium is used for the enrichment step in bacteriophage isolation and why? | DSPB (Deca strength phage broth [10X stronger than regular media]). It allows for the growth of E.coli |
| What culture is used for the plaque assay? | E. Coli |
| In the plaque assay, E. coli not only acts as a host, but it also _______? | selects only for phage that infects E. coli so the number of coliphage will be increased |
| The DSPB is used to ______? | increase the number of E. coli, which INdirectly increases the number of phage |
| DSPB is not broth for phage because ____? | Phage cannot grow in media without a host (the host in this experiment is E. coli) |
| For the plaque assay, why did the sample have to be centrifuged (using vortex mixer) prior to filtration? | To remove all the cellular debris so that the membrane of E.coli didn't get cloged |
| For the filtration step in the plaque assay, the centrifuged enrich sewage was filtered using a ____________? | Nalgene filtration apparatus |
| What kind of agar plates were used for the filtration step of the plaque assay? | EMB (Eosin Methylene Blue) agar |
| In the filtration step of plaque assay, for EMB plates, if lactose and sucrose are both fermented, then _______? | The whole colony is purple |
| In the filtration step of plaque assay, for EMB plates, if only lactose is fermented, then ______? | Only the center of the colony is purple |
| What is Eosin in relation to the plaque assay? | An acidic dye which gives coliforms (E. coli in this case) a green metallic sheen |
| ___________ is used to prepare an overlay of the E. coli and phage on top of a TSA plate | Soft Nutrient Agar |
| For the seeding step in the plaque assay, why were the TSA plates pre-warmed in the incubator? | To prevent the Soft Nutrient Agar from solidifying as soon as it hit the plates, which allowed the soft nutrient agar to flow over the surface of the TSA plate |
| After the seeding step of plaque assay, plaques appeared on the plates within? | 2-2.5 hours |
| For the plaque assay (seeding), longer periods of incubation will cause the plaques to ________? | Become confluent (combined together), making it difficult to count the individual plaques |
| What is the result of gram staining klebsiella pneumoniae? | Pink color (Gram -) |
| What is the result of the capsule stain of klebsiella pneumoniae? | Cells were purple, background was pink and capsule was present around cells |
| What is the result of the MacConkey agar test for klebsiella pneumoniae? | Red in color from neutral red (Lactose fermenter, acid producer) |
| What is the result of the EMB agar test for klebsiella pneumoniae? | Dark purple color (Fecal coliform) |
| What is the result of the gram stain for staphylococcus epidermidis? | Purple in color (Gram +) |
| What is the result of the capsule stain for staphylococcus epidermidis? | Purple cocci clusters, pink background, NO capsule present |
| What is the result of the endospore stain for staphylococcus epidermidis? | Pink cocci clusters with NO green endospores |
| What is the result of acid fast stain for staphylococcus epidermidis? | Blue cocci clusters (NON-acid fast) |
| What is the result of the PEA agar test for staphylococcus epidermidis? | Growth occurs (PEA allows growth of G+) |
| What is the result of the MSA agar test for staphylococcus epidermidis? | Pink growth (pH of 8.4+, Non-mannitol fermenter, non-pathogenic) |
| What is the result of the MacConkey agar test for staphylococcus epidermidis? | No growth (Non-lactose fermenter [MacConkey inhibits G+]) |
| What is the result of the endospore stain for bacillus stearothermophilus? | Pink bacilli with presence of green endospores (G+) |
| What is the result of the endospore stain for clostridium sporogenes? | Pink bacilli with presence of green endospores (G+) |
| What is the result of the acid fast stain for mycobacterium smegmatis? | Pink bacilli chains (Acid fast) |
| What is the result of the gram stain for pseudomonas aeruginosa? | Pink bacilli (Gram -) |
| What is the result of the flagella test for spirillum volutans? | Spirochete with flagella on each end (amphitrichous) |
| What is the result of the flagella test for pseduomonas fluorescens? | Bacilli with flagella tufts on one end (lophotrichous) |
| What is the result of the flagella test for proteus mirabilis? | Bacilli with flagella all around (peritrichous) |
| What is the result of the PEA agar test for proteus mirabilis? | No growth in PEA (G-) |
| What is the result of the MSA agar test for E.coli? | No growth (non-pathogenic, non-halotolerant) |
| What is the result of the HE agar test for E.coli? | Yellow-salmon color (sugar fermenter) |
| What is the result of the PEA agar test for enterococcus faecalis? | Growth (G+) |
| What is the result of the HE agar test for enterococcus faecalis? | No growth (G+ inhibited) |
| What is the result of the MSA agar test for staphylococcus aureus? | Yellow color (acid producer, pathogenic, mannitol fermenter) |
| What is the result of the EMB agar test for staphylococcus aureus? | No growth (G+ inhibited) |
| What is the result of the EMB agar test for proteus vulgaris? | Pink growth (slow fermenter, low acid producer) |
| What is the result of the MSA agar test for proteus vulgaris? | Colorless (non-lactose fermenter) |
| What is the result of the HE agar test for salmonella typhimurium? | Black precipitate (sulfer reducer) |
| What is the purpose of PEA agar? | To isolate Streptococci and Staphylococci from mixed cultures |
| What is the purpose of MSA agar? | To isolate and differentiate pathogenic organisms like Staphylococcus aureus |
| What is the purpose of the MacConkey agar? | To select and differentiate member of Enterobacteriaceae |
| What is the purpose of the EMB agar? | To isolate fecal coliforms |
| What is the purpose of the HE agar? | To isolate Salmonella and Shigella species from other enterics |
| What acts as the mordant for acid fast? | Heat and steam (help drive stain into cell) |
| Flagellin are arranged into a _______ structure | helical |
| Bacteria that do not move with flagella (non-motile) exhibit __________ | brownian motion |
| The genus spirillum tends to have ________ flagella | amphitrichous |
| the genus proteus tends to have _______ flagella | peritrichous |
| the genus pseudomonas tends to have ______ or _____ flagella | Monotrichous or lophotrichous (fluorescens was monotrichous) |
| What are the advantages of wet mount and hanging drop? | can readily determine the presence or absence of motility (no incubation time required), can determine cell morphology (shape) and arrangement, no heating is required so there is no distortion |
| What is the advantage of motility test media | Can be used to detect motility of pathogens |
| What is the minimum requirements for steam sterilization for 1 L of media? | 15 minutes at 121.6*C (250*F) and 15 psi |
| Serratia marcescens produces a red pigment at 25-30*c called | prodigiosin |
| Micrococcus luteus appears yellow due to _______ | carotenoid pigments |
| How does PEA inhibit gram negative bacteria? | phenyl ethyl alcohol hinders their DNA synthesis |
| PEA is used to grow _________ | anaerobes if 5% sheep blood is added |
| What bacteria genus are included in Enterobacteriaceae (gram -)? | Salmonella, proteus, E.coli, klebsiella, serratia |
| What are fecal coliforms? | Rod-shaped, gram negative, non-sporulating, facultative anaerobic bacteria |
| A microscope that allows light rays to pass through a slide/specimen and then through various lenses to the eye. The lenses magnify the object. | brightfield |
| the measure of a lens’ ability to capture light coming from a specimen and use it to make the image; increases with the use of oil and the higher objectives | Numerical aperature |
| Limit of resolution (D) = ? | wavelength / NA condenser + NA objective lens |
| How are basic dyes able to penetrate and stain bacteria? | They contain chromophores with positive charges, which penetrate the negative bacteria |
| what are the three functions of heat fixing? | To kill the bacteria, To fix them to the slide, To coagulate cytoplasmic proteins to make cells more visible |
Created by:
JHaddad