Download Microbiology Lab Final Exam. and more Exams Advanced Education in PDF only on Docsity!
Microbiology Lab Final Exam.
What type of media would you use to allow growth of many bacterial species from the sampled environment? - Answer-TSA (Tryptic soy agar) Where would you incubate a soil sample? - Answer25 degree celsius incubator Where would you incubate a mouth/touch plate? - Answer37 degree incubator because it's the human body temp Where would you incubate a sample from the frozen tundra? - Answerin a fridge thats about 4 degrees celsius??? Resolution definition - AnswerThe ability to distinguish two small objects apart from each other. Magnification definition - AnswerCalculated by multiplying the mag. of the objective lens by the mag. of the eyepiece.
- eyepiece=10X
- scanning-objective=4X
- low power objective=10X
- high dry objective=40X
- oil-immersion objective=100X
Parfocal definition - Answer-when you focus a specimen under low mag. you can switch the objective to a higher mag. and your specimen will be almost in focus...just use fine focus Proper handling of microscope - Answer-after use all oil must be wiped off the objective and all other parts of the microscope with lens paper
- always store microscope with the lowest power objective pointing down. How to use immersion oil - Answer-add one drop of immersion oil to the slide
- add oil when switch from 40X lens to 100X lens
- none of the other objective lenses should get oil on them
- never switch back to a low power objective once you add oil to the slide STUDY MICROSCOPE DIAGRAM - Answer Purpose of simple stain - Answer-Make otherwise transparent microorganisms visible under microscope
- Stains make it easy to determine morphology of bacterium General stain chemical properties - Answer-Most commonly used stains are basic
- positively charged, so bind electrostatically to negatively charged surface of bacterial cell
- Acid stains bind to less common positively charged molecules, used for special purposes
- ex. CV, safranin, methylene blue
Purpose of heat-fixing after the smear has air-dried - AnswerHeat fixation glues the bacteria onto the slide.
- if still wet, would just burn water off
- too much heat can damage cell walls and distort shape of organism KNOW COLONY MORPHOLOGY DESCRIPTIVE TERMS - Answer Purpose of streak plate - Answer-To isolate colonies using pure culture technique What zone do you expect the most and the least colonies? - AnswerMost: The first area inoculated on the plate (zone 1) Least: The areas that were inoculated last (zones 4 and 5) Why do you flame your loop before streaking the next zone on the plate? - Answer-to kill any bacteria remaining on the loop so that bacteria from zone one will only be spread to zone 2 and then only bacteria from zone 2 will be spread to zone 3 and so on...
- results in dilution of the inoculum A gram stain... - Answer-Discovered by Hans Christian Gram
- Differentiates bacteria based on thickness of peptidoglycan layer (not the presence/absence of OM) Gram positive - Answer-ex. Staphylococcus aureus
- has thick cell wall (thick pg layer) that retains crystal violet-iodine complex during staining procedure
- stain dark purple Gram negative - Answerex. E. coli
- have thin layer of pg and do not retain the crystal violet-iodine complex
- stained by counterstain, safranin and appear pink
- pink Gram stain procedure - Answer1. make smear
- add 2 drops crystal violet-let sit for 2 min
- Rinse excess stain off with water at angle, shake off excess water, dont need to dry
- Add 2 drops Gram's iodine for 1 minute
- Add 2-3 drops of decolorizer at angle until purple color no longer runs off slide, shouldnt need more than 5 drops, removes excess CV
- Rinse with water, shake off
- Add 2 drops safranin for 30 seconds
- rinse with water, blot with bibulous paper FUNCTION of GRAM STAIN REAGENTS - Answer Spore stain purpose - Answer-used to view endospores produced by Gram positive bacteria ex. B. subtilis
- Cell is stained red, endospore is stained green
- Malachite green is responsible for green spore color
- Safranin is used to stain the rest of the cell
What are capsules? - Answer-extracellular structures produced by bacteria that allow them to adhere to surfaces, evade the human immune system, and protect cells from predation. What is the acid-fast stain used for? - Answer-Used for bacteria containing waxy lipids (mycolic acids) in their cell wall
- Ex. M. tuberculosis What are mycolic acids - Answer-waxy lipids in the cell wall of Mycobacterium
- allow the cells to be highly resistant to dessication What color are acid fast and non-acid fast bacteria after staining? - Answer- Acid fast=red
- Non-acid fast=blue ex. Staph aureus What stains are used in the process resulting in the different outcomes? - Answer1. Carbol fuchsin (thick layer of mycolic acids prevents decolorization of this primary stain by acid alcohol) so referred to acid fast
- Methylene blue counterstain stains non-acid fast bacteria blue Steps in acid fast stain - Answer1. Stain with carbol fuchsin ---both AF+/- will be red
- Decolorize ---AF+=red still; AF-=no color
- Counterstain with Methylene blue ---AF+=piink still; AF-=blue
Amount of powder required to prepare TSA - Answer40g/L or 40g/1000mL
- given volume needed
- set up ratios and cross multiply Standard autoclave conditions - Answer121 degrees celsius 15 minutes 15 psi How would you know if the medium was contaminated before inoculating it with your cultures? - Answergrowth? Bacterial culture medium - Answer-an artificial substrate used to support the growth of bacteria in the lab
- supplies all essential macronutrients (C, N, O, H, P, S), micronutrients (Mg, Fe, K, Ca, Zn, etc.) and growth factors Defined medium - Answernutrients are added to the medium in the form of pure chemicals Complex medium - Answernutrients are added to the medium in the form of animal/plant extracts What does agar do? - AnswerIt doesnt change the nutritional composition of the medium, it is added solely as a solidifying agent.
- it's liquid at 100 degrees and solidifies at 44 degrees celsius
- differentiates between lactose fermenters (pink colonies) and non- fermenters (no color change) Mannitol Salt Agar (MSA) SELECTIVE - Answer-selective
- high salt concentration
- selects for Staphylococcus species Mannitol Salt agar (MSA) DIFFERENTIAL normal color=pink - Answer-differential
- mannitol
- differentiates between mannitol fermenters (yellow colonies) and non- fermenters (no color change) Phenylethyl alcohol agar (PEA) - Answer-selective
- phenylethyl alcohol
- inhibits growth of Gram-Negative bacteria Eosin methylene blue agar (EMB) SELECTIVE - Answer-selective
- Eosin Y, methylene blue
- inhibit growth of Gram-Positive bacteria Eosin methylene blue agar (EMB)
DIFFERENTIAL - Answer-differential
- lactose
- Differentiates between strong lactose fermenters (green metallic colonies), weak lactose fermenters (pink colonies) and non-fermenters (no color change) Eosin methylene blue agar (EMB) ENRICHMENT - Answer-enrichment
- lactose, sucrose
- enhances growth of fecal coliforms Blood Agar ENRICHMENT - Answer-enrichment
- blood
- Enhances growth of fastidious bacteria (pathogens) Blood Agar DIFFERENTIAL - Answer-differential
- blood
- Differentiates based on type of hemolysis. alpha-hemolysis (partial destruction of RBCs, greenish color may develop around colonies), Beta- hemolysis (complete destruction of RBCs, zone of clearance surrounds colony), and Gamma-hemolysis (non-hemolytic) Catalase test, What is catalase? - Answer-catalase is an enzyme produced by bacteria to detoxify hydrogen peroxide to water and oxygen gas. Hydrogen is
Purpose of Durham tube - Answer-some fermenters produce CO2 gas in addition to acidic by-products
- inverted Durham tube traps produced gas Carbohydrate fermenter: - Answer-acidic by products produced (lactic acid, acetic acid) will turn the medium yellow Non carbohydrate fermenter: - Answer-medium will turn pink due to alkaline by-products produced by utilization of peptone
- no gas in tube Citrate test
- media used, what are you testing for?, Expected pos/neg results - Answer- tests the ability of organisms to utilize citrate as a carbon source
- requires enzyme citrate permease, a transport proteins
- organisms on citrate convert ammonim dihydrogen phosphate (nitrogen source) to ammonium and ammonium hydroxide
- ammonium products from citrate utilization change pH from neutral to alkaline
- pH indicator=bromthymol blue, green at neutral pH, so green medium when uninoculated --positive test, citrate producer=growth with deep blue color --negative result, non citrate utilizer=little to no growth and no color change Motility test - Answer-medium is semi-solid and contains the indicator tetrazolium chloride (TTC)
- allows differentiation of motile and non-motile bacteria
- organisms growing reduce colorless TTC to red colored formazan
- Has lower agar concentration so that motile bacteria can swim
- inoculation=stab with needle --positive test=red, diffuse growth radiating from stab line --negative test=red growth only along stab line Triple sugar iron agar (TSIA) - Answer- TSIA is a differential medium that diffs. organsims based on ability to reduce sulfur and ferment carbohydrates.
- contains 3 sugars: lactose, sucrose, glucose; ferrous sulfate, and phenol red pH indicator
- beef extract, yeast extract, peptone (C and N sources), sodium thiosulfate (source of reducible sulfur)
- the iron ferrous sulfate is hydrogen sulfide indicator
- shallow agar slant with deep butt to provide aerobic and aneaerobic environ.
- stab and streak Yellow slant/yellow butt - Answerglucose and lactose and/ or sucrose fermentation with acid accumulation in slant and butt Red slant/yellow butt - Answer-glucose fermentation with acid production.
- Proteins catabolized aerobically in slant with alkaline products Red slant/red butt - AnswerNo fermentation.
- Proteins catabolized aerobically and anaerobically with alkaline products Red slant/ no change in butt - AnswerNo fermentation
- pH of 4.4 and below=red Nitrate reduction test - Answer-nitrate broth used to test ability to reduce nitrate (NO3) to nitrite (NO2) during anaerobic respiration
- contains nutrients and potassium nitrate
- AFTER INCUBATION:
- add sulfanilic acid and alpha-naphthylamine --IF: after incubation and addition of sulfanilic and a-naph. red compound forms, positive for nitrate reduction --IF: after incub. and add. of sulfanilic acid and a-naph. the medium doesnt turn red, the organism may not be able to reduce nitrate or the organism was able to denitrify the nitrate to nitrite to produce ammonia or Nitrogen....SO ADD some powdered zinc... IF turns red after zinc=negative result IF doesnt turn red after zinc+ positive test Viable counts - Answer1mL/10mL= 10^- 1 100 microL= 0.1 mL 1 mL= 10^-3 L DF - Answer-dilution factor, no units DF= (volume added in mL/total volume in mL) X DF of previous tube FDF - Answer-final dilution factor of countable plate, units= mL FDF=(volume plated in mL) x (DF)
OCD - Answer-original cell density, units are CFU/mL OCD= CFU/FDF CFU= # colonies
- always has positive exponent Biofilms vs planktonic cells - Answer-in lab bacteria grown in liquid culture float around freely in the medium= planktonic cells
- In nature, bacteria often attach to a surface and form a biofilm: the growth of a microbial community on a surface 5 stages of biofilm development - Answer1. Reversible attachment = cells attach and detach from the surface
- Irreversible attachment = cell irreversible attaches to surface via cell adhesion structures like pili
- Maturation I
- Maturation II = during 3 and 4, the biofilm becomes encased in an extracellular matrix aka slime
- Release = some of the cells within the biofilm are released and are planktoic and can remain free or attach elsewhere to start new biofilm Examples of biofilms - Answer-In nature= slimy rocks next to river coated w/ biofilms, film on shower door, ring in toilet
- In healthcare setting= danger. once biofilm established and encased in extracellular matrix, the cells within the biofilm exhibit increased resistance to antibiotic therapy and antimicrobial agents. ex. Indwelling catheters, surgical implants, contact lenses and cases if not cleaned routinely and may cause eye infections if biofilms form
Missense mutation - Answer Nonsense mutation - Answer Gradient plate composition for spontaneous antibiotic resistance mutant lab - AnswerAntibiotic containing agar overlaid on antibiotic-free media
- both layers angled to dry at slants
- creates antibiotic concentration gradient because antibiotic diffuses into lower agar layer with amount of diffusion proportional to thickness of the agar layer
- top layer=TSA + streptomycin
- bottom layer= pink TSA
- more pink side is low antibiotic concentration Lac+ vs lac- growth patterns on MAC - Answer-Lac+ is the wild-type E. coli strain, has ability to ferment lactose....grows as pink colonies
- Lac- is the mutant E. coli and after UV exposure may find some lac- cells.... growing as colorless/white colonies Why did we use a UV lamp to generate lac- E. coli? - Answereasy to handle it's the physical mutagen that induces mutation to make lac- E. coli bla - Answer-B-lactamase gene
- product of this gene provides resistance to ampicillin.
- B-lactamase is an extracellular enzyme that acts on the B-lactam ring structure found in ampicillin gfp - Answer-gene encoding for the green fluorescent protein derived from the jellyfish Aequorea victoria araC - Answer-gene encoding the AraC regulator that is derived from the arabinose catabolism operon
- in the presence of arabinose, araC interacts w/ the sugar and binds to the promoter region upstream of arabinose catabolism genes so organism can utilize the sugar... BUT the pGLO plasmid only has the regulator and the gfp gene that replaced the genes for arabinose catabolism.
- Binding of arabinose-activated AraC results in activation of gfp gene expression oriT - Answerorigin of plasmid replication What is natural competence? - Answer-when a bacteria can undergo transformation (take up naked DNA from the environment on its own)
- involves the transfer of linear DNA ex. Streptococcus pneumonia What is artificial competence? - Answer-induced competence cant take up DNA on it's own
- ex. E. coli
- repeated washes in presence of high levels of CaCl2 (100mM) folllowed by a quick heat shock treatment (42 degrees for 5o seconds)
