microbe - lab final review

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Things in purple- unsure Things in red is unanswered Practical review slides Which tube is called a transport media? C What is tube B used for? Motility, O2 requirement What ingredient is tube A lacking? Agar What is the name of this organism? Schistosoma mansoni (class Trematodes phylum platyhelminth) What is the sexual status of this organism? Dioecious
What category does it belong to? Trematodes (flukes) What is the name of this test? Carbohydrate fermentation - Durham test Which tube indicates a positive result? Tube A Note: Bacteria metabolize sugar which results in acidic waste products. If the tubes in the picture are testing for lactose, which one is the potential pathogen? B What does the pink color indicate? Tube B no carbohydrate fermentation. Note: bacteria metabolize proteins resulting in neutral waste product What Gram reaction is shown here? Gram negative bacteria bacilli What antibiotics would you expect this organism to be resistant to? Penicillin or any cell wall inhibitors - narrow spectrum What morphology is shown in the image above? Rod shape. Bacillus
What is the name of this microbe? Trichomonas vaginalis What disease does this microbe cause? Trichomoniasis / vaginitis What is this microbe’s method of motility? Visible flagella What phylum does it belong to? Archaezoa What method of isolation is this? Spread plate Can you determine a concentration from this method? Yes if you know the dilution factor of the plate culture - serial dilution If there are 132 colonies on this plate and the dilution was 10-6, what was the original concentration? 132 x 10 6 = 1.32x10 8 bacteria per mL
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What structure is shown here? Capsule Was this slide heat fixed? No Give a possible genus and species name for this organism? Streptococcus mutans What is the name of this media? Blood agar Green - alpha Yellow - beta n/a - gamma How would you describe this reaction? Alpha hemolysis partial breakdown What is the genus and species name of this organism?
Streptococcus pneumoniae What is the genus name of this organism? Aspergillus What name is given to this spore-bearing structure? Conidia (naked/exposed spore) What is the name of this test? Indole Test - tests for breakdown of tryptophan, tryptophan is amino acid Which tube is a positive result? The pink band positive for breakdown of tryptophan (MV test) What is the reagent for this test? Kovac’s reagent add 4 drops after incubation Is this test part of the IMViC? Yes
amino acid tryptophan broken down Which of the white blood cells in this view is the most common and what is its function? Neutrophil - function is phagocytosis Basophil is the one to the far left - histamine releaser (allergies) Which of the white blood cells in this view is less common and what is its function? Lymphocyte - specific immunity
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What name is given to object B? Brewer’s jar (no oxygen) obligate anaerobe, facultative anaerobe, and aerotolerant can grow in Jar B Describe the environment created in object A? Low oxygen high CO2 ( candle reduce oxygen in jar) What kind of organism would grow the same in both of these objects? Facultative anaerobes, and aerotolerant but aerotolerant grows exactly the same in both (prefers oxygen environment but can switch to fermentation in no/reduce oxygen ) -Facultative anaerobe, aerotolerant, obligate aerobe can grow in Jar A What enzyme is being tested for? Urease (organism produce exoenzyme urease which breaks down urea into ammonia and changes pH to alkaline ) urease test breaks down urea into ammonia Which tube indicates a positive result? Pink bacteria can produce urease breaking down ammonia What does a positive result indicate? Presence of Enterobacteriaceae (gut flora and urogenital bacteria)
What is the name of this microbe? Giardia lamblia What disease does this microbe cause? diarrhea What is this microbe’s method of motility? Flagella (visible) Which phylum does it belong to? Archaezoa What is the name of this media? MacConkey plate (selective gram negative, differential lactose fermenter (pink growth)) What is indicated by the color in A? Gram negative bacteria positive for lactose fermentation Why is there no growth in B?
No growth on B indicate a gram positive bacteria If growth is seen on B it is lactose fermenter negative Non lactose fermenter is pathogenic What structure is demonstrated by this staining technique? Endospore What is the general age of this culture? old Give a possible genus and species name for this organism. Bacillus subtilis Boiling and pasteurization time takes longer, for boiling is 10 min, pasteurization is 30 min
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What is the genus name of this organism? Rhizopus What name is given to this spore-bearing structure? Sporangium (encased spore) When spore is in a sac we call it a sporangium What enzyme is causing the bubble to form? Catalase What is the reagent for this reaction? Add 2 - 3 drops of Hydrogen peroxide H2O2 Which are possibly Streptococcus? A and B are positive, C and D are negative for catalase production.
Streptococcus are catalase negative Staphylococcus are catalase positive What is the name of this organism? Clonorchis sinensis (liver fluke) How does this organism reproduce? Monoecious - both male and female reproductive organs in the same individual What is the name of this test? Methyl-Red (MR) Used to detect acidic waste produce of glucose fermentation Which tube indicates a positive result? Red broth,
What does a positive MR result indicate? Acidic waste from glucose fermentation (positive red) What does a positive VP result indicate? Use glycol metabolic pathway to breakdown glucose (positive red) Neutral waste produce Both tests cannot be positive, but both can be negative What is the name of this organism(s)? Paramecium spp. What’s going on here? Asexual reproduction (binary fission) What is this microbe’s method of motility? Move using cilia (think hair like fiber) Is this activity sexual or asexual? Asexual
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What is the name of this media? Mannitol Salt Agar (MSA ) What is indicated by the yellow color? Positive mannitol fermentation , halophile Give the genus and species name of the bacteria from the skin that ferments mannitol? Staphylococcus aureus - halophile and mannitol fermenter - yellow result Selective medium – halophiles salt loving bacteria Differential - mannitol fermenters What is the name of this microbe?
Plasmodium spp. What disease does this microbe cause? Malaria What is this microbe’s method of motility? No motility (is inside red blood cells) What phylum does it belong to? Apicomplexa What is the name of this instrument? Calibrated loop , used for UTI/ urine How much inoculum does it hold? 0.001 mL or 1uL or 10^-3mL If there are 78 colonies on a plate inoculated with this loop, what was the original concentration? 78 x10 3 =7.8x10 4 bacteria per mL. Not considered UTI/ UTI is >100,000 TSI test Which tube indicates only glucose was fermented? The pink slant and yellow butt indicate only glucose was fermented. What does an all yellow, slant and butt, indicate? All yellow slant and butt - positive for glucose fermentation and positive for lactose and/or sucrose fermentation
Yellow butt - glucose fermentation only Yellow slant - positive lactose and/or sucrose fermentation Pink slant and butt - negative carbohydrate fermentation Black butt - positive for H2S gas production Why is the last tube all pink? Negative for carbohydrate fermentation What is the name of this microbe? Balantidium coli What disease does this microbe cause? diarrhea What is this microbe’s method of motility? Cilia What phylum does this microbe belong to? Ciliata
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What method of isolation is this? Streak, cannot be used to count concentration, purpose for streak is to isolate pure colonies Can you determine a concentration from this method? No What is this method of isolation used for? Isolating pure bacteria culture What is the name of this media? EMB plate , selective for gram negative, differential for lactose fermenter (B & C) What is the genus and species of C? E. coli - rapid lactose fermenter Which organisms might be pathogens?
Pathogens D (non lactose fermenter) C and B positive for lactose fermenter D is gram negative but not lactose fermenter D, is pathogenic C is E. Coli What is the name of this test? Kirby-Bauer Test, no zone of inhibition means resistance What is the name of this medium? Mueller-Hinton plates Is the bacteria tested here resistant or susceptible to most of the agents being tested? Susceptible Penicillin is G+, with no zone of inhibition bacteria is gram negative Polymyxin is G- , with no zone of inhibition bacteria is gram positive
Name the gas produced here. H2S hydrogen sulfide How was this medium inoculated? Slant - inoculated with a loop Butt - inoculated with a needle The agar is stabbed through the center to the bottom of the tube and the agar slant is streaked and incubated (Triple Sugar Iron Agar aka TSI) What is the name of this test?
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Starch Hydrolysis Test, if starch is the only nutrient you will have no growth with bacteria that doesn't contain starch (Bacteria on the right side) Which colonies are positive? Clear zone around the bacteria (left) What does a positive result indicate? The breakdown of starch through the production of enzyme amylase What kind of enzyme is amylase Digestive enzyme What is the name of this creature? Taenia pisiformis How does it reproduce? Monoecious - both male and female reproductive organs in the same individual
What is the name of this test? Citrate Slant test Which tube indicates a positive result? Blue What does a positive result indicate Formation of citrate What is the genus name of this organism? Microscopic Fungi (penicillium) What name is given to this spore-bearing structure? Conidia (naked spore)
What is the name of this test? Litmus milk test E. sugar fermentation, if white at bottom extreme fermentation B. protein breakdown, if white is shown its litmus reduction E is acidic A. complete peptization Which tube indicates an acidic response? Pink tube indicates acidic as sugar is fermented What does the brownish clear liquid in A indicate? Peptonization (complete protein breakdown) What is the name of this media?
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Blood agar plate What is indicated by the clear areas? Beta hemolysis - complete breakdown of RBCs - golden appearance What organism from a throat swab is identified by this reaction? Streptococcus pyogenes (Strep throat) What is the name of this microbe? Entamoeba histolytica What disease does this microbe cause? Diarrhea What is this microbe’s method of motility Pseudopodia What is the TDT here?
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10 minute Does this represent boiling or pasteurization? Boiling - kills all vegetative cells in 10 mins Does this represent old B. subtilis or young B. subtilis? Young B.subtilis Practical Study guide I. Metabolic Tests ● Fermentation (Durham tubes) wk 7 Test for fermentation of one carbohydrate (glucose or lactose) Yellow - Acidic (+) for carbohydrate fermentation Pink - Basic (-) for carbohydrate fermentation; protein breakdown Red/Orange - neutral - uninoculated (lack of turbidity) Gas Bubble - fermentation resulted in gas production ● Urease wk 7 Test for production of enzyme urease (converts urea into ammonia) Yellow slant - (-) for enzyme urease Pink slant - (+) for enzyme urease (breakdown of urea into ammonia) ● Indole wk7 Test for the breakdown of tryptophan (amino acid) into indole Add 4 drops of Kovac’s reagent AFTER incubation Yellow - (-) for the breakdown of tryptophan Pink - (+) for the breakdown of tryptophan
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● Catalase wk 8 Test for production of enzyme catalase Distinguishes between Strep. (-) and Staph. (+) Add 2-3 drops of H2O2 Positive - bubbles ● MR---VP wk7 Only one test can be positive MR or VP Both can be negative MR and VP MR: acidic waste VP: neutral waste Add 5 drops of methyl red Add 10 drops reagent A and 5 drops reagent B (wait 45 mins) Red broth (+) Red broth (+) Yellow broth (-) Yellow broth (-) ● Starch Test for the production of enzyme amylase - digest starch Add iodine directly into starch plate after incubation Iodine + Starch = purple/black complex (-) starch was not broken down Clearing zone around bacteria =(+) fir amylase - starch digested No clearing zone =(-) fir amylase - starch still present
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● Citrate wk7 Test for use of citrate as a sole carbon and energy source Green slant (-) for used of citrate not capable Blue slant (+) for use of citrate ● Nitrate reduction wk8 Testing for reduction of NO3 Requires 3 drops of Reagent A and 3 drops of Reagent B If no color change add a pinch of zinc with a wooden applicator stick -Pink after reagent A/B = Nitrate reduced to nitrite (+) it gained electrons -Pink after zinc = Nitrate was NOT reduced (-) -No color change after zinc = Nitrate reduced to NO or N2 gas (+) y
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● Litmus milk wk 8 ● TSI II. Specialized media ● EMB Selective - Gram negative bacteria Differential - lactose fermenters No growth - Gram positive bacteria Growth - Gram negative bacteria Pink growth - Lactose fermenter Metallic green growth - Rapid lactose fermenter (E.coli) *If not pink or green it is not capable of fermenting lactose
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● Sabouraud (SabDex) wk 2 Classification - Selective media Purpose - Encourages the growth of certain microbes & prevents others from growing Unique Traits - Low pH, High sugar - Grows fungi ● MSA (Mannitol Salt Agar) - selective and differential media commonly used for identification of pathogenic Staphylococci -Only halophiles (salt loving) microbes are capable of growing on this media -Red-pink to yellow = growth indicating a halophile and positive for mannitol fermentation -Red-pink = growth indicating a halophile but negative for mannitol fermentation ● Thioglycollate
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● MacConkey- is specifically used for isolation of gram negative Enterobacteriaceae - Gram negative lactose fermenters appear as = pink colonies/ pink media - Gram negative, non-lactose, fermenters appear = unchanged/colorless ● Snyder’s agar Differential for fermentation Green = negative Yellow = positive ● Blood ● Sucrose agar ● Mueller---Hinton III. Growth Characteristics ● Streak plate: used to isolate pure cultures in the form of single colonies (clones) Colonies of different bacterial species can be differentiated based on color, texture and size ● Spread plate used to count bacterial colonies to estimate the total number of bacteria per mL in a broth culture.The broth culture may need to be diluted through a process known as serial dilution IV. Equipment ● Microscope Use of microscopes Parfocal: when one lens is in focus all other lenses will also be in focus Objective lenses: 4X: scanning lens 10X:low power lens; start with this lens 40X:high power lens; ONLY use fine focus knob 100X:oil immersion lens; Use immersion oil and ONLY use fine focus knob Total magnification (TM):ocular lens X objective lens= (10X) What would be the TM at 40X objective? =400X ● Different forms of media (broth/slant/deep)
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Broth - liquid agar - grow large quantities of bacteria - loop Slant - solid agar - transport & storage - loop Deep - semi solid agar - Test for O2 requirements and/or motility - needle Plate - solid agar - quantify or isolate bacteria - loop Reducing media (thioglycolate broth) -Contains sodium thioglycolate chemically reacts with and eliminates oxygen present in the media -Inoculated using loop -Tubes are NOT shaken or mixed rolled between palms for a few seconds and then incubated -Location of bacterial growth in the tube indicates oxygen requirement ● Oxygen requirements A.Obligate aerobe:REQUIRE oxygen B.Obligate anaerobe:grow ONLY in the absence of oxygen C. Aerotolerant anaerobe:not harmed by oxygen but do not use it either D. Facultative anaerobe:can use oxygen but can also grow in the absence of oxygen ● Candle jar: capnophilic environment (low O2, high CO2) ● Brewer’s jar:anaerobic environment (no O2) ● Incubator:same levels of O2 as the atmosphere ● broth/slant/deep) ● Calibrated loop, Needle hold s exactly 1uL ● . Micropipettes V. Procedures ● Dilution series ● Gel electrophoresis ● Counting microbes ● Unknowns (using dichotomous key) ● Disk diffusion / Kirby---Bauer ● Oxygen requirements ● UV light Damages DNA by forming thymine dimers, where two side by side thymines get stuck together Long exposure = more killing (direct) Most distance = less killing (indirect0 Exposure to visible light = less killing (indirect) ● Temperature control (TDT, TDP) ● Isolation of bacteria
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VI. Microscope slides ● Protozoa ○ Balantidium (phylum ciliata) move using cilia Bean shaped or bar shaped nucleus Cysts ingested from contaminated food/ water Causes diarrhea ○ Giardia (phylum archaezoa) move using flagella (visible) -Giardia lamblia Tear drop shape, two nuclei, and multiple flagella hanging off Causes diarrhea ○ Paramecium - (phylum ciliata) move using cilia -free living -sexual reproduction: conjugation -asexual reproduction:binary fission ○ Trypanosoma (phylum euglenozoa) move using flagella (not visible) Trypanosoma brucei S shaped squiggles among RBCs Causes African sleeping sickness (neurological condition)
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○ Entamoeba (phylum amoebozoa) move using pseudopodia -Entamoeba histolytica Oval shaped Nucleus looks like a ring with a dot inside Causes diarrhea ○ Euglena (phylum euglenazoa) move using flagella (not visible) Euglena spp. Thin torpedo shaped organisms Free living and photosynthetic ○ Amoeba proteus(phylum amoebozoa) move using pseudopodia Visible pseudopods (finger like projections) Free living ○ Plasmodium (phylum apicomplexa) no motility Plasmodium spp. Ring like organism Inside RBCs Causes malaria
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○ Trichomonas (phylum archaezoa) move using flagella (visible) -Trichomonas vaginalis Big round organism with a dense nucleus and flagella hanging off Causes trichomoniasis/ vaginitis ● Fungi ○ Mold slide (Penicillium, Aspergillus, Rhizopus) Rhizopus- Spore bearing structure = sporangium (spores encased in an outer covering) Penicillium Aspergillus Spore bearing structure = conidia (naked/ exposed spores)
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○ Helminths- platyhelminthes = flat worms/ Nematodes = round worms Sexual Status: Monoecious:both male and female reproductive organs in the same individual Dioecious:male and female are separate individuals ○ Schistosoma - Schistosoma mansoni (platyhelminth--trematodes) ○ Trichinella Trichinella spiralis - Nematodes ○ Taenia (platyhelminths -- cestodes) ○ Clonorchis - Clonorchis sinensis-liver fluke (platyhelminth--trematodes)
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● Leukocytes: neutrophils, eosinophils, basophils, lymphocytes, monocytes VII. Stains ● Direct/simple : heat fix basic dye(+) (methylene blue, crystal violet, safranin) Advantage: can use multiple dyes, can view slides multiple times Disadvantage: heat fixing causes some shape and size distortion. ● Acid---fast wk5 distinguishes bacteria on presence or absence of mycolic acid in cell wall. Positive = looks red/purple has mycolic acid in cell wall/waxy layer Negative = looks blue with no mycolic acid in cell wall Primary stain: carbol fuchsin cover smear w/dye and place on beaker of hot water Mordant: heat - keep slide on beaker for 5 min Decolorizer : acid alcohol 5-10 sec quick rinse, rinse with DI H2O 2nd stain : methylene blue 45 sec, right with DI H2O ● Negative : NO heat fixing Acidic dye (-) (nigrosin) - reples dye stains backgroung Advantage: view true morphology (shape) & size of bacteria Disadvantage: slide can only be viewed once ● Capsule combination of direct stain and negative stain Stain slide (nigrosin or india ink) NO heat fix (air dry 5 min.) Stain cell (crystal violet) Rinse (DI H2O) Blot dry (capsule appears with clear halos against dark background & stain cells) ● Gram distinguishes bacteria on the thickness of peptidoglycan Positive : purple thick peptidoglycan Negative : pink thick peptidoglycan Primary stain: crystal violet Mordant: iodine Decolorizer : ethanol 2nd stain : safranin ● Endospore Primary stain: malachite green cover smear w/dye and place on beaker of hot water Mordant: heat keep slide on beaker of hot water for 5 min - makes dye penetrate/stick Decolorizer : DI H2O rinse well - challenges the cell to keep/loose dye 2nd stain : safranin 45 sec - rinse with DI H2O
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VIII. Specific microbes ● Beta---hemolytic ● Acid---fast ● Halophile, mannitol fermenter ● Endospore Green spores will appear upon heat application - old ● Metallic green colonies IX. Microbes in water ● Presumptive test (MPN, multiple tube test) Lactose Durham tubes are used to determine MPN (most probable number) ● Confirmatory test EMB plate to check for Gram negative lactose fermenters ● Completed test Gram stain to confirm that the cells are Gram negative bacilli X. Urine analysis ● Quantitative analysis Determine the number of bacteria per mL of urine UTI >100,000 bac/mL 10,000 - 100,000 bac/mL false positive and negative for UTI False positive Patient did not wipe properly - did not get a mid-stream catch - sample was not tested in a timely manner by the lab ● Qualitative analysis Examine the features of bacteria that grow in urine XI. Miscellaneous ● Categories of disinfectants & chemotherapeutics ● UV repair mechanisms Endonuclease: slow & inefficient BUT active 24/7 (dark or light) DNA photolyase: fast and efficient BUT only active under visible light Human possess - Endonuclease ● Lysozyme You do NOT need to review Generation time and N(f) equations.
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