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by: kirkmathews

Microlab_Final.pdf BIOL 2042


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This is a 100% guide to getting an A on the final! All the information asked on the final study guide is included into this 22 page monster of a document, full of pictures to make memorizing which ...
Microbiology Lab
Dr. Kunz
Study Guide
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This 22 page Study Guide was uploaded by kirkmathews on Saturday April 23, 2016. The Study Guide belongs to BIOL 2042 at University of North Texas taught by Dr. Kunz in Spring 2016. Since its upload, it has received 33 views. For similar materials see Microbiology Lab in Biology at University of North Texas.


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Date Created: 04/23/16
--------------------------------------------Fungi-------------------------------------------- How do you classify fungus? By their spores Mycology- the study of fungi Fungus have sexual reproduction, while bacteria have none, and are non motile The media used to grow fungus is called Sabouraud dextrose – contains Glucose, has lower pH and has organic matter Rhizopus nigricans – Non septate in stalk with a big bulb Asexual spore - sporangiospore Plate – notice black white fuzz with black ring ^Rhizopus nigricans <Aspergillis niger V Aspergillus – Segmentation in stalk - Bulb stays brown when stained - Grown on Sabouraud agar medium Macromophology - Front looks like black mold, back looks like vascular mountains* Lacto-Phenol Cotton Blue Stain Used to stain chitin in cell wall of fungi such as; Sacchromyces cerevisae Rhizopus nigricans Aspergillus Penicillium Chitin- makes up the cell wall of fungus Fungi Characteristics- Non-motile, non-photosynthetic, chemoheterotrophic Zygomycota, Basidiomycota, Ascomycota, Imperfect Fungi Lichen- Mutualism between fungus and either alga or cyanobacteria Fungus give structure, Cyanobacteria provides carbohydrates via photosynthesis < Penicilium -Grows on orange peel, common fruit mold Fusarium -Identify the macroconidia (banana looking thing) ^ Penicillium macroscopic morphology Saccharomyces cerevisiae - Yeast - Budding - A drop added to Lactophenol Blue Test in order to stain - -How do Yeast make a living? - Chemoheterotroph - Eat dead organisms and decaying material -------------------------------------------Algae------------------------------------------- How do you classify algae? By color and DNA sequencing All algae are in kingdom protists Phycology- study of seaweed and algae How do we classify algae? By color Chlamydomonas Green Alga -Have flagella Spirogyra -Green Alga Spirals inside of ridged wall Volvox -Green Alga Diatoms -Gold Alga Glassy cell wall made of Cillica Desmid – Bilateral symmetry --------------------------------------Cyanobacteria--------------------------- Cyanobacteria (Prokaryotic)– contain chlorophyll a, phycoerythrin -used to conduct oxygenic photosynthesis Have a membrane sac like structure – Thylakoid Anabaena- produces heterocyst that fixes nitrogen Heterocyst- large, transparent, thick-walled cell found in the filaments of certain blue-green algae and fungi Gleocapsa- Oscillatoria- Look like stacks of pancakes It moves in an oscillating manner Spirulina- Spirals not inside of ridged wall ------------------------------------Protozoa----------------------------------- Protozoan – Cell wall is called a pellicle Two subgroups of flagellated Protozoans Archaeozoa – flagellated with no mitochondria Eugleazoa – flagellated with mitochondria Paramecium- move by cillia Ciliated Vortichella- partially non motile when they find a host Have cillia Amoeba- Use Pseudopods to eat and move Euglena- Move by Flagella Trypanosoma -move by flagella African sleeping sickness Larger and outside RBC Plasmodium(Malaria) Non-motile Members of Sporozoa Trophozoite is the active vegetative stage -growing stage The infectious stage is called the sporozoite -Absorbing nutrients from host Trichomonas vaginalis -have five flagella that allow movement -STD -----------------------------------Antagonism-------------------------------------------- Be able to identify Streptomyces griseus filamentous gram-positive rod stain Antagonism – involves the production of a substance such as an antibiotic by one member of the association. This substance is detrimental to the second member of the association. Soil is the most common place these organisms are found Members of the genus Streptomyces are responsible for half of the antibiotics in the market Streptomyces bacteria are filamentous gram positive rods Produces colored conidiospores at the tips of hyphae and produces geosmin (the odor of soil) In this experiment, we used water agar because water has no nutrients and is slightly selective by not allowing growth for anything else Extras— Which one has a cyst stage? Why is that important? ----------------------------Lab 29 – Bacteriophage------------------------- Viruses- noncellular, obligate intracellular parasites -can contain DNA (Virus) or RNA (Retrovirus) as genetic material Capsid-Protein coat for DNA/RNA - Tail fibers are present which have the ability to attach to specific sites Receptors-sites on host cell that virus attaches to -Generally proteins or glycoproteins on surface of cell Bacteriophage- Viruses that infect bacteria  Coliphage- Virus that specifically infects E. coli Lytic phage- Causes lysis of the host cell by taking over replicative machinery of the cell and then using these resources for creation of the components for new phage Once the host cell is depleted of resources, the host cell bursts and phage particles are released to infect other bacterial cells -Produces Clear zones of clearing (Plaques) due to killing of all surrounding bacteria Lysogeny- Much less common, phage incorporates into the genome of the host at a specific site. Lysogenic bacteria grow and replicate normally for any number of generations, but during times of cellular stress, the phage can remove itself from the genome and then enter the lytic cycle. This “incorporation” relationship is known as lysogen. -Produces Cloudy zones of clearings (Plaques) due to growth of lysogens Plaques- Zone of clearing, each one is produces by a single viral particle or “plaque-forming unit” (PFU) Titer – number of bacteriophage in a sample (PFU/DF) Purpose of this Lab- in order to see viruses infect E.coli and produce plaques What was happening to the cells during the 10 minute incubation time at 37*C? -The viruses were infecting the E.coli Why were additional E.Coli cells added to the phage mixture after first incubation? -To create a lawn ---------------------------------Lab 23- Tests---------------------------------- Bacteriological examination of water - E.coli and coliforms Coliform: group of organisms recognized as the principal indicator of unsanitary conditions (soil, intestine, plants, and environment). Facultative anaerobic, gram-negative, lactose-fermenting rods, such as Citrobacter, Klebsiella and Enterobacter, are phenotypically similar to E coli, the principal indicator organism of fecal contamination. ----------------------------------------Direct measurement----------------------------------------- directly counting bacteria on a slide or by using dry weight in order to calculate cell mass. Direct measurement does not discriminate between living and dead cells. Must have 30-300 colonies on a plate in order to count Dry weight- should be used with filamentous organisms such as Streptomyces and fungi. Turbidity- can be measured using a spectrophotometer, because turbidity is proportional to number of bacteria in a solution, usually at 540 nanometers (More bacteria, more things to block light, more cloudy) ------------------------------------Methylene blue reduction test------------------------------- When added to milk, aerobic bacteria absorb all of the oxygen and Methylene blue is used as the final electron acceptor, and then when reduced turns white The rate at which Methylene blue turns white is proportional to the number of bacteria/ml Older something is = more bacteria Lab 24 E. coli is found in the human body, and therefore is human waste. If this is found in water, water is contaminated with human waste. E. coli – Grouped as a coliform organism, which is a group of gram-negative, lactose fermenting, facultative anaerobe organisms -----------------------------------------Membrane filtration---------------------------------------- recognized by US Public Health Services for the detection of E. coli and other coliforms in water. It is a quantitative test and has the advantage of detection and enumeration. Advantage- you can test large volumes of water -0.45 micrometer pores -after filtering water, place membrane filter onto a media such as Eosin Methylene Blue or MacConkey + MUG agar plates -Eosin Methylene Blue – -E. coli shows a metallic green sheen -Purpose is to distinguish E. coli from coliforms -Indicator- The dye responds to a change in pH, going from colorless to black under acidic conditions -Inhibitor- The dye methylene blue in the medium inhibits the growth of gram-positive bacteria Positive- E. coli will have a metallic green sheen Negative- Gram negative bacteria (including coliforms) will grow colorless colonies < Eosin Methylene Blue MacConkey + Mug > We saw this in vials -MacConkey + MUG detects E.coli based on presence of β-glucoronidase enzyme that fluoresce under UV light at 355 nanometers. ---------------------------------------Colisure (IDEXX) Test---------------------------------------- Purpose of Colisure Test- This test is for detection and confirmation of total coliforms and E. coli in water. Advantages of Colisure Test- It’s very quick, taking only 4 hours to conduct a test. This is a Presence/Absence test, and it’s qualitative. This method is based on Defined Substrate Technology. It is based on the fact that when coliforms metabolize the nutrient indicators, that they produce a color/fluoresce. Coliforms-Chromogenic substrate- β galactosidase enzyme E. coli- flourogenic substrate- β glucoronidase enzyme This test can detect bacteria at 1 CFU/100 ml at 24 hours Results of Colisure Test Negative – yellow/gold Positive for coliforms- Red/Magenta Positive for E. coli- Red/Magenta and Fluoresces at 355 nanometers (UV Light) -Fecal contamination indicator -------------------------------Most Probable Number-------------------------------- Definition and Purpose- Statistical method to estimate number of viable microorganisms in a sample. This method is based on the probability of a single cell that is present in a given amount of the sample. Benefit- Good to use for counting bacteria present in very small numbers Indicator- Brilliant Green Bile Lactose Broth (BGBLB) - selective medium for detection and enumeration of E. coli and coliforms based on the fermentation of lactose and gas production (in a Durham tube). Positive – Yellow solution Negative – Green solution This is a presumptive test for coliforms, after the experiment is done, streak an Eosin methylene blue agar plate with the highest three dilutions that were positive. Coliforms – purple/red colonies upon growth with a red zone around them or metallic green sheen color Further testing is needed to verify specific organism. ------------------------------Compact Dry E.coli count------------------------------- Purpose- Isolation and Enumeration of coliform/E.coli Advantages- Only require a very small volume (1ml) Indicators- Pink-purple colonies are indicative of coliforms and blue-colored colonies are indicative of Escherichia coli. Chromogenic substrates- Magenta Gal (5-bromo- 6-chloro-3-indoxyl-beta-D-galactopyranoside) X-Gluc (5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt) Procedure- The growth area is 20cm2. The total count can be obtained by multiplying the average number of colonies observed in one 1cm x 1cm square grid by 20. Average out each 1 cm by 1 cm square then multiply by 20. Instructor notes: Membrane filtration- large volume samples (e.g. City water), quantitative, EMB media (Eosin methylene blue), It’s a differentia media, coliforms turn pink, E.coli metallic blue sheen, filter is .45 microns, direct count. Compact dry/E.coli- small volume (1ml), quantitative, regular bacteria are white, E.coli are blue, coliforms are pink/purple MPN (most probable number) - statistical, very small amounts of bacteria are detectable, BGBLB media (in tubes) (brilliant green bile lactose broth), also has Durham that collects gas that E.coli ferments, yellow tube = positive Colisure- quick only a few hours (4), qualitative, tests for E.coli and other coliforms and non coliforms, positive test is purple (has coliforms), yellow test is negative, and E.coli containing tubes are fluorescent under UV black light. This is caused by MUG + β-glucoronidase = fluorescent We want (0) E.coli in our water --------------------Bacterial Count in Food and Milk------------------------------- Importance of bacterial count in milk- Milk is pasteurized to a temperature that ensures the killing of all pathogens, but can still contain bacteria and coliforms. You don’t want to drink bacteria. -Grade of Milk – The higher the grade of milk, the less total bacteria are included Importance of bacterial count in meat- Generally, the older meat is, the more bacteria it has. Why do we have to do dilutions? –In order to get an accurate reliable count of bacteria per ml. Serial Dilutions- Ten fold dilution 1:10 (Original:Total) 1 ml into 9 ml of sterile water -Hundred fold dilution 1:100 0.1 ml into 9.9 ml of sterile water Colony Forming Units (CFU/ml)- Each colony is counted on a plate, and divided by the diluting factor (in milliliters) an accurate count is a plate with a count of 30-300 CFUs. 0 3 Scientific notation- 1x10 = 1 Stock solution 1x10 = 1,000 Viable Cell Number --------------------------Lab 27- Transformation------------------------------------- Fred Griffith- In 1928 observed that non-virulent Streptococcus became virulent when injected into mice along with a head-killed virulent strain of Streptococcus. Only virulent bacteria were recovered from the dead mice. Transformation- change of non-virulent bacteria into virulent bacteria, cells up taking naked DNA Competent- Cells which are able to take in naked DNA from environment Strains used in this experiment Acinetobacter ADP1 & Acinetobacter ADP6 ADP1- Has enzyme protocatechuate oxygenase so capable of degrading p-hydroxybenzoate (POB) as carbon source. Also was lysed. ADP6- mutant strain which lacks the enzyme protocatechuate oxygenase, an enzyme used in degradation of POB. Also was transformed. -ADP6 will grow on minimal media containing glucose, but cannot grow when POB is main carbon source TENS solution – Used to lyse the cells T-Trisbuffer- Used as a pH buffer to maintain DNA E- ethylenediamine acid (EDTA)- used as chelating agent which removed divalent cations that are required by DNA degrading enzymes (prevent DNA degradation) N- sodium hydroxide (NaOH)- Breaks cell wall S- sodium dodecyl sufate (SDS)- Detergent that breaks cell membrane After lysing ADP1 cells and mixing with ADP6, the ADP6 cells that are competent for protocatechuate oxygenase (enzyme used for POB catabolism) will grow 2 genus that are naturally competent are- Acetobacter and Streptococcus Utilization of P-hydroxybenzoate is a catabolic reaction E.coli can be made artificially competent using Magnesium chloride or calcium chloride Media used- Glucose minimal media- Both ADP1 and ADP6 could grow on this, used as a control to make sure both strains were alive POB (para-hydroxy benzoate)- The ADP1 and transformed ADP6 grew on this because they had the enzyme protocatechuate oxygenase ------------------------------------Lab 28 – Conjugation------------------------------- Conjugation- unidirectional transfer of genetic material that requires cell to cell contact, within same species -Requires presence of a conjugable plasmid Plasmid- Closed-circular, extrachromosomal DNA capable of self-replication -contains tra genes which are necessary to transfer the plasmid from the donor to the recipient The transfer of plasmid occurs while the donor and recipient cells are close together. The donor cell holds the recipient cell with a Pilus. Pilus- The pilus proteins are encoded on the plasmid and form a rod-like structure on the surface of the donor cell. The plasmid replicates and is transferred -When conjugation is complete, both the donor and recipient have copies of the plasmid. The organisms observed are two strains of Pseudomonas putida Leucine – an amino acid required for growth (growth factor) TOL plasmid- conjugable plasmid which contains genes for the catabolism of m-toluate. Auxotroph- lacks the ability to synthesize a growth factor Prototroph- has the ability to synthesize a growth factor Phenotype- physical traits displayed by organism, codes such as Leu + being prototrophic for leucine P. putida PAW15- Le– Tol+ leucineauxotroph, contains the TOL plasmid -Will grow on m-toluate but only with the addition of Leucine -Donor strain P. putida 503CA- L+u Tol- leucine prototroph, does not contain TOL plasmid -Will not grow on m-toluate as the sole carbon source -Will grow fine on glucose minimal medium P. putida Daughter strand- +eTol + leucine prototroph, contains the TOL plasmid -Will grow on m-tolulate OR glucose minimum medium as sole carbon source --------------------------------------Lab 31 – IMVC------------------------------------- Indole test - used to differentiate between enteric bacteria which are able to split indole from the tryptophan molecule (E. coli, Proteus vulgaris, Morganella and Edwardsiella) from those that cannot. Adding Kovac’s solution (4 drops) will form a red-ring if the solution is Indole positive. Methyl Red Test (Mixed Acid Fermentation)- The primary purpose of the Methyl Red Test is to differentiate between E. coli (MR +) and Enterobacter (MR -). E. coli is a mixed acid fermenter, producing lactic acid, acetic, formic, and succinic acids, as well as ethanol in the process of fermenting glucose. Adding Methyl Red (4 drops) is simply an indication of a change in the pH of the solution. If the bacteria produce produce mixed acids during fermentation, then the medium turns red. If the bacteria do not produce mixed acids, then the media turns yellow. Red- 4-5 pH Yellow-6-7 pH Voges-Proskauer Test (Butanediol Fermentation)- The primary purpose of Voges-Proskauer Test is to differentiate between organisms that produce Butanediol and mixed acid fermenters. Fermenters use one pathway or the other, so this test is differentiative for Enterobacteriacae. -Glucose is catabolized, fermenting Acetoin and Butanediol. After addition of the KOH (14 drops) and (14 drops of α-nepthol), Acetoin turns into Diacetyl, and Diacetyl mixes with the Creatine (Also found in KOH) to produce a Red color. Brown/Red near top- Positive Citrate - Citrate is an intermediate in the krebs cycle. Some bacteria are capable of using this as a carbon source (Enterobacter, Salmonella, Serratia, Klebsiella). Blue- Positive Green- Negative ------------------------Lab 32 – Sugar Fermentations------------------------------ Glucose Fermentation : The fermentation of Glucose is an anaerobic process in which the glucose is broken down to acids and other gaseous byproducts. The production of acids causes the pH indicator (phenol red) to change from a red color to a yellow color. The entrapment of bubbles in the inverted Durham tube inserted in the broth tube is an indication that gases have been produced. Yellow + Gas- positive Red-negative Lactose is essentially the same just using Lactose as a carbon source. -----------------------------Lab 33 – Motility------------------------------------------- Prokaryotic movement- Most occur by flagellar movement Eukaryotic movement- May possess cilia or a different type of flagellum than that of a bacterium Benefits of SIM Test- Save time and money Sulfide Test – Is used to detect the ability of an organism to produce 2 S from the reduction of sulfur in the SIM medium to sulfide. Motility Test - is an indication of an organism’s ability to move away from the inoculation site. --------------------------Lab 13.4 – Oxidase Test------------------------------------- Use a cotton swab on a well isolated colony from a fresh bacterial culture Add one drop of oxidase reagent to the sample on the cotton swab Look for color changes to dark purple within 5-10 seconds. Disregard color changes after 60 seconds. Only organism that we dealt with that had a positive result – Psuedomonas fluorescens ----------------------------Lab 34 – Other Biochemical Tests---------------------- Starch Hydrolysis - Test to see if organism produces the enzyme Amylase which breaks down starches into simple sugars. -Amylose is broken down to Dextrin using Amylase, which is then broken down again to Maltose using Amylase, then again to D-glucose, using Amylase. In order to detect the organisms’ ability to metabolize starch on an agar plate with starch added as the carbon source, an indicator such as Iodine. Is used. In areas where the organism has catabolized the starch, Iodine will not stain. In the areas where organisms have not catabolized the starch, Iodine will stain. Black Plate with clear halo around streak- Amylase positive Black Plate with no clear spots – Amylase negative Urea - Test to see if organism has the enzyme Urease that breaks down urea to carbon dioxide, water, and ammonia. This test is used to differentiate the Proteus group from other Gram pathogens such as E. coli. Proteus is an opportunistic pathogen responsible for urinary tract infections. The indicator is Phenol Red, which turns bright pink at high pH. Positive - Bright Pink/Ammonia’s pH in positive solution- 8.1 Negative - Orange/Urea starting pH – 6.8 --------------------------------GRAM + TESTS--------------------------------------- MSA (mannitol salt agar)- Indicator- Staphylococcus saprophyticus ferments mannitol which drops pH and turns it yellow Inhibitor- 7.5-10% salt content in agar Positive- Yellow Staphylococcus saprophyticus (media turns yellow) Negative- Red Staphylococcus epidermidis (media stays red) Catalase- The enzyme used to break down toxic Hydrogen Peroxide (H O ) into2 2ygen (gas)and Water (H2O) Reagent used- Hydrogen Peroxide S. epidermidis Most bubbles S. saprophyticus Most bubbles B. megaterium Little bubbles E. faecium No bubbles Starch hydrolysis - α-amylase breaks down starch. If starch is left intact + iodine turns media fully brown then the organism lacks the Amylase enzyme. If after adding iodine there is a halo around the streak, then the organism has the α-amylase enzyme. This is testing for B.Meg which breaks down starch (+) and Staph Epi Which doesn’t break downstarch(-). After Iodine is added, Left Positive Right Negative Bile esculin - has esculin, ferric citrate and 5% bile. Identifies group D Streptococci (Enterococcus) Bile inhibits growth of non group D organisms. If Enterococcus is present, esculin is hydrolyzed to form dextrose and esculetin. Esculetin will react with Ferric ions to turn the media black. Positive- Dark black Negative- Light Brown -----------Lab 35 – Identification of Enterobacteriacaea (Enteropluri) ---- Purpose of the Enteropluri- Multimedia tube used for the identification of Enterbacter. It is a self- contained compartmented plastic tube containing 12 different convientional media, and 15 biochemical tests. What two characteristics must organisms have in order to use the Enteropluri for their identification? -Gram negative Rod and Oxidase Negative ---------------------------------------Epidemiology-------------------------------------- Principal of the Experiment- Explain how disease is transmitted in a population Epidemiology- Science that deals with when and where diseases occur, how they are transmitted to the human population and who is spreading the disease Epidemiologist-Scientists that study and find answers to the when, where, and how. Epidemic- When a disease spreads throughout a population of people in a short time(The United States) Pandemic- Spread of a given disease worldwide Communicable diseases- Diseases that spread through contact or through vectors (such as lice, mosquitos, etc) Organisms used in this experiment- -Pathogen- Serratia marcescens – used as dummy pathogen because it will turn red when incubated at room temperature -Normal microbiota- Micrococcus luteus What is the function of the Center for Disease Control and Prevention? -It is the duty of the scientists and epidemiologists at the CDC to track trends and analyze new and reemerging infectious disease issues around the world. -What is a MMWR? – The Morbidity and Mortality Weekly Report is a weekly epidemiological digest for the United States published by the Centers for Disease Control and Prevention (CDC). Essentially a report discussing how where and how certain diseases are found, and how frequently. ---------------------------------Microbial Symbiosis----------------------------------- Plant-microbe interactions- Bacteria sometimes infect plants in order to receive nutrients in exchange for some kind of benefit (Mutualism) Symbiosis- Rhizobium infects the root of a leguminous plant in order to provide the plant nitrogen in exchange for nutrients Enzymes involved- I don’t know you tell me guy ---------------ID items used in all experiments done in lab--------------------- Micropipetters (Know how to read them, page 78) Just know all measurements are in Microliters (µl) ^Microcentrifuge Tube


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