BIO-205L Midterm Exam Study Guide

Identify the guidelines for safety in the laboratory?
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Identify the guidelines for safety in the laboratory?
-Wear a lab coat and goggles
-Wear closed-toed shoes
-Tie back long hair
-Know where the fire extinguisher is located
-Know where the eyewash is located
-Clean lab benches before and after lab with 10% bleach
-Wash hands before and after lab
-When working with live cultures
•Carry them in a test tube rack
•If a culture spills, cover with paper towels and soak with bleach
-Do not eat or drink in the laboratory
-Do not put on makeup in the lab (e.g. lip balm, contact lenses)
-Discard anything containing or touching a live organism into biohazard trash
-All glass goes in the glass biohazard trash
Why do we use 10% bleach in the lab?
It is used to disinfect benches before and after work, and when a spill occurs. Kills microbes.
What is ubiquity?
Microorganisms are ubiquitous, which means they exist everywhere.
What are fomites?
Are microbial reservoirs or sources that play an active role in the spread of diseases.
What are nosocomial infections? Examples?
Hospital-acquired infection. Examples- UTI, MRSA, respiratory pneumonia
What is bacterial colony?
A cluster of closely packed cells that are growing on a agar surface
Why are agar media plates inverted when placed in the incubator or refrigerator?
To prevent condensation from forming and dripping onto the bacteria.
Ocular lens?
Eyepiece of a microscope; magnifies the image (10x).
Specimen stage?
A movable platform where slides can be placed for observation.
Condensor lens?
Concentrates the light and makes illumination of the specimen more uniform (reduces light loss).
Iris diaphragm?A mechanical device on top of the condenser that can be opened or closed with a lever and regulates the light column illuminating the specimen.Objective lensSelection of lens options that provide different magnification.Scanning power objective lens?Magnifies about 4x.Low power objective lens?Magnifies about 10x.High- and dry- power objective lens?Magnifies about 40x.High power oil immersion objective lens?Magnifies about 100x.Light source?Electrically powered bulb at the base of the microscope that provides illumination to the specimens / CONTROLS AMOUNT OF LIGHT.Knob?Controls intensity of light.Light microscopy?Light passes through specimen, then through series of magnifying lenses-magnification and resolution-condenser lens.Magnification?The ability of a lens to enlarge an object-ocular lens: 10x-objective lens: 4x, 10x, 40x, 100x.Total magnification?Product of the magnifying power of the ocular lens and objective lens.Resolution?Ability of lens to show detail smallest distance between 2 points on a specimen that can still be distinguished as 2 separate entities.Resolving power?Maximum distance between 2 objects where those objects still appear separate -the ability of a lens to resolve 2 independent points as distinct entities -dependent on the wavelength of light used.Simple Microscope?Has only one lens.Compound Microscope?Has 2 sets of magnifying lenses combined to enlarge objects.Par focal?The capacity of an instrument to maintain focus regardless of the magnification used.What are the 2 factors that enhance resolution?Wavelength of light used & numerical aperture.Why is oil used with the 100X objective?-light refracts/ bends as it moves from glass to air -oil bridges the gap between the glass slide and lens and reduces refraction -oil has nearly the same refractive index as glassBiological stain?Color added to enhance the view of a biological slide.Simple vs. differential staining?Simple stain: uses a single dye-shows cell morphology (shape & arrangement). Differential stain: uses more than one dye, differentiates structure of cell wall.Principle of positive and negative staining?positive stain: dye sticks to the specimen negative stain: dye sticks to backgroundThree types of bacterial morphology (shape and arrangement)?coccus (circular) bacillus (rod-shaped) spirillum (spiral)What information is obtained from a simple stain?Cell shape, size, and arrangement.How to make smears on glass slides from broth?- flame loop and let cool - place a drop of water on the slide - remove cap of broth culture and pass through flame - get a loopful of culture from the broth - pass mouth of tube through flame and recap - thinly spread culture on the slide and let air dry - heat fix the slideHow to make smears on glass slides from slant cultures?With sterile loop transfer 2-3 loopfuls of water onto glass slide, sterilize, transfer a pinpoint amount of culture from agar to drop of water, tap several times with loop to dissolve, spread thin, air dry, heat fix.Purpose of the following when making bacterial slides: -clean glass slide -thin uniform smear -air drying -heat fixing-clean glass slide: to prevent cross-contamination -thin uniform smear: to ensure bacteria is clearly visible with no clumps -air drying: to prevent burning of bacteria during heat fixing -heat fixing: it adheres the smear to the slide and allows the sample to more readily take up stainsList and describe the rationale for the steps required to prepare a bacterial smear?1. Place a drop of water into the wax circle that has been created on the slide. 2. Heat fix the loop and obtain a very small sample of the bacterial colony. 3. Gently mix the bacteria into the water drop. 4. Let the sample air dry. 5. Heat fix the sample to kill the bacteria, firmly attach the smear to the microscope slide, allow the sample to more readily take up the stain.Describe the morphology and cellular arrangement of two species of bacteria?Bacillus subtilis = bacillus (rod shaped), chains (string like) Streptococcus mutans= coccus, single cellsState two reasons why a negative stain is used?to observe bacterial shape and to examine the presence of capsulesDiscuss the implications of capsules in bacterial diseases?-found mostly in gram-negative bacteria -the limit the ability of phagocytes to engulf the bacteria -prevents the direct access of lysosome contents with the bacterial cell, preventing their killingCan a negative stain show the internal structures of a bacterial cell?No, because the background is colored and the cell is clear.As you rush to be in time for the Microbiology lab, you accidentally spill coffee on your lab coat and the white fabric gets stained. Is this a biological stain or simply a compound that is capable of imparting color to the fabric? Explain your reasoning?It is simply a compound stain capable of imparting color. A biological stain refers to a compound that changes the color of features of a cell such as cell walls or the nucleus of a cell and helps to view them more clearly. A coffee stain does not do that.Name four positive stains and two negative stains and explain how their chemical charge affects staining of bacteria?positive: stain blue because of their thick peptidoglycan walls -streptococcus pyogenes negative: stain red because of their thin peptidoglycan wall -methicillin- resistant staph aureus -nigrosin and eosinCompare and contrast the staining characteristics of three species of bacteria and bacteria found in teeth scrapings using positive and negative staining techniques?bacillis subtilis=white strep mutans= white mouth organisms= white w/black ring Gram positive stain purple/blue Gram negative stain pink/redGram staining with special emphasis on: · Primary stain · Mordent · Decolorizing agent · Secondary/counterstain · Differences in cell wall: gram(-) and gram(+)Primary stain- crystal violetAll cells take up the purple crystal violet stain - stain for 1 min(if negative, primary stain is lost during decolorization) Mordent- Iodine; keeps crystal violet in gram positive cell wall Decolorizing agent- alcohol; 95% ethanol used to remove crystal violet from gram-negative cells Secondary/counterstain- safranin; used to give red color to gram-negative cells Differences in cell wall: gram(-) and gram(+)- plasma membrane surrounded by thin peptidoglycan layer and second outer membrane containing lipopolysacharides (LPS) and lipoproteinsplasma membrane surrounded by thick peptidoglycan layer containing teichoid acidsList the Gram staining steps in order?Heat Fix 1. Primary stain- Crystal violet (1 minute) Wash off stain with di-water 2. Mordant- Gram's Iodine (1 minute) Wash off the iodine 3. Decolorization- Acetone-alcohol (hold slide at 45 degree angle and apply decolorizer, do this until it runs clear) Stop decolorization by washing slide with gentle stream of di-water 4.Counterstain- Safranin (1 minute) Wash off gently for only a few seconds 5. Blot dry with bibulous paper & air dry 6.Examine slide under oil immersionWhich is the most crucial step in the Gram staining procedure? Explain?Decolorizing so that we can see the different parts of the bacteria clearly.What would be the appearance of bacteria if: · You forget to counterstain with safranin · You use methylene blue instead of safranin · The mordant is not used · The decolorizing step was too long, too short, or omitted?-no counterstain: gram-positive would appear purple and gram-negative would be colorless -methylene blue: you'd see dark purple in gram-positive and blue in gram-negative -no mordant: it wouldn't be bright and gram-positive might appear pink -decolorizing step is messed up: under colorizing will not completely remove the crystal violet causing the gram-negative to appear gram-positivePrinciple of differential staining?Series of dyes and additional chemicals to stain bacteria in contrasting colors. To see the difference in bacterial cell wall structuresWhy is differential staining advantageous over simple staining?The differential staining determines the shape, arrangement, and gram reaction while the simple staining only determines the morphology.Gram's stain helps observe which specific bacterial cell anatomy?Divide into gram-positive and negative based on their cell wall.What is Gram-variability?Some organisms undergo changes in their cell surface chemistry (and Gram reaction) as they get older; gram-positive cells that may appear gram-negative using the gram stain procedure.Describe three conditions that may result in Gram-positive organism staining Gram-negative?1) the method and techniques used 2) the age of the culture 3) the organism itselfWhat is the medical importance for a Gram stain?It can determine the presence of bacteria in a sample, as well as differentiate between the two distinct bacteria species, gram + and gram - -can help clinicians choose the appropriate antibiotic for treatment.List two Gram-positive bacteria and their diseases?-mycobacterium tuberculosis: tuberculosis -mycobacterium leprae: leprosyList two Gram-negative bacteria and their diseases?-E. coli: food poisoning -vibrio cholorae: choleraWhich one is more difficult to treat─ an infection caused by Gram-negative bacteria or by Gram-positive bacteria? Explain?Gram-negative because they have a second outer layer made of lipids and carbs.On a Friday evening, the health care personnel on duty forgot to refrigerate the urine sample and left it on the counter. On Monday, the sample was sent to the lab and the Gram stain showed great variability ranging from intense purple to shades of red. How would you account for this result?Bacteria that are not kept under proper holding conditions or that are over 18-24 hours old tend to show immense amounts of variability, not identifying as either game-negative or gram-positive.Acid-fast stain with special emphasis on: -Primary stain -Decolorizing agent -Secondary/counterstain -Differences in cell wall: Acid-fast and Non-Acid-fastPrimary stain- carbol fuchsin; red stain used to penetrate mycolic acid present on outer cell wall Decolorizing agent- acid-alcohol; mixture of ethanol and hydrochloric acid Secondary/counterstain- methylene blue; gives acid-fast negative cells a blue color Differences in cell wall- acid-fast: surrounded by layer of mycolic acid. nonacid-fast: no mycolic acidWhy is heat or phenol used with the application of primary stain during acid-fast staining?Drives stain into waxy layer so it stays.Why is acid-alcohol used instead of 95 % ethyl alcohol for decolorizing the primary stain?Acid fast organisms have a waxy lipid layer that does not stain easily and ethyl alcohol would decolorize all of the stain.Which specific bacterial anatomy is observed with the acid-fast stain?Used to confirm presence of mycobacteria in cell wall -mycolic acidWhat is the diagnostic value of the acid-fast stain?Used to confirm the presence of mycobacteria, leprosy (m. leprae), and TB (m. tuberculosis).Identify two acid-fast positive and two acid-fast negative bacteria and describe the method and steps used in an acid-fast stain?StepsAllow smear to air-dry and then heat fix in the usual manner. Place the slide with smear side up on the staining rack in the chemical fume hood. Cover the smear with a square piece of bibulous paper, and apply carbol fuschin onto the paper. Allow the cells to stain for 10 minutes. Remove the paper by using forceps, and discard it into the biohazard bag. Wash the slide gently with tap water to remove stain. Decolorize with acid-alcohol, adding the reagent drop-by-drop until the alcohol runs almost clear with a slight red tinge. Wash with tap water. Counterstain with methylene blue for 2 minutes. Wash smear with tap water. Blot the slide dry with bibulous paper, and examine slide under oil immersion. Dispose of staining solution in the properly labeled waste container, and place the glass slides in the glass waste container. positive: mycobacterium (has mycolic acid layer) mycobacterium leprae and bacillus anthratis negative: NONEIdentify the unique molecule found in the cell wall of an acid-fast positive bacterium and explain why this bacterial genus is difficult to stain?Mycolic acid layer- waxy type of fatty acid that requires strong phenolic chemical dyes combined with heat to drive stain into it, once stained it cannot be removed.Endospore stain with special emphasis on: · Primary stain · Secondary/counterstain · spore vs. vegetative cell-Primary stain- malachite green; used to penetrate the spore coat and give green color to endospores -Secondary/counterstain- safranin; gives red color to vegetative cells -Spore vs. vegetative- spore are resistant to heat, dessication, chemicals, and radiation vegetative cells are cells that are actually growing, rather than forming sporesExplain the result if: if heat was not applied during application of the primary stain?No heat: stain wont penetrate endospore cells.What is the purpose of the boiling/steaming step in the endospore staining process?To drive the stain into the spore coat.Is bacterial sporulation a reproductive process? Explain?No, the bacteria simply encases itself into a "resting" state, in which all of its contents are encased in an endospore to remain viable under extremely harsh conditions. IT IS A SURVIVAL MECHANISM.Which specific bacterial anatomy is observed with the Endospore stain?Endospore vs. vegetative cell.Why is it called the Christmas tree stain?Endospores stain GREEN and vegetative cells stain RED.List two spore-forming bacteria and their diseases?-Bacillus anthracis: anthrax -Bacillus cereus: food poisoningDescribe the method and steps of an endospore stain and illustrate the staining characteristics of endospores and vegetative cells?-Place a slide over a beaker of boiling water and flood smear with malachite green for 10 minutes -Wash off with DI water -Cover with safranin for 2 minutes -Rinse and blot and allow to air dry -Endospores: green Vegetative cells: redIdentify three bacterial species that form endospores and describe the disease caused by each one?-Bacillus anthracis (causes anthrax) -Clostridium tetani (tetanus) -Bacillus cereus (food poisoning)Identify the distinguishing chemical found in the coat of endospores and explain its significance in the resistance of endospores to various chemicals/treatments?Have an outer layer of heat/chemical resistant molecule called dipicolinic acidCulture medium?Nutrients prepared for microbial growth.Nutrient agar slants?Used to maintain stock cultures so bacteria can grow on its surface.Nutrient broth tubes?Used to grow large numbers of organisms.Nutrient Agar Plates?Used to isolate pure cultures of organisms and to do plate counts.Bunsen burner/Bacti-cinerator?Bunsen burner: used to provide heat/flame when needed in the lab-used to sterilize test tube openings and all other tools necessary to prevent cross contamination. Bacti-cinerator: used to sterilize inoculating loops. sterilizes microorganisms with infrared heat.Inoculating wire loop/ needle?Long handled instrument used to dispense small amounts of sample onto surface.Contamination?Process by which unwanted microbes are accidentally introduced.Cross-contamination?When something is not properly sterilized and is used on a different bacteria type (cross of bacterias).Sterilization?The process that completely destroys all microbial life, including spores.Aseptic techniques?Techniques that prevent contamination by unwanted microorganisms.Inoculation?Process of introducing microorganisms into a sterile culture medium.Inoculum?The cells used to start a microbial culture.Bacterial culture?The microbial cell growth on nutrient media.Turbidity?Cloudy appearance of nutrient solution in a test tube due to growth of microbe population.Pure culture?population of microbial cells derived from single cell -contains only one type of microbe.Mixed culture?The population that contains more than one type of microbe.Sub culture?Transfer of microorganisms from one medium to another to prepare fresh microbial cultures.Streak for isolation?A technique for isolating colonies to generate pure culture of bacteriaFlaming the inoculating loop/wire before and after each inoculation?To sterilize and prevent cross contamination.Holding the test tube caps in the hand (pinky finger)?To allow for quick/ easy access to put the cap back on and to prevent it from getting contaminated by touching other surfaces.Cooling the inoculating loop/wire before obtaining the inoculum?To prevent burning/killing the bacteria and making them unusable.Flaming the mouth of the test tube immediately after uncapping and again before recapping.To prevent contamination.Define: antibiotics, bactericidal vs. bacteriostatic?Antibiotics- A medicine (such as penicillin or its derivatives) that inhibits the growth of or destroys microorganisms Bactericidal- Antimicrobial effect that result in microbial death are called bactericidal agents Bacteriostatic- Those causing temporary inhibition of growth are bacteriostatic agentsHow can you tell whether the test microorganism was sensitive or resistant to the chemotherapeutic agent?Sensitive-zone of inhibition Resistant-no zone of inhibitionWhat does the zone of inhibition represent?Degree of sensitivity of bacteria to a drug/antiseptic.State the factors that influence the size of the zone of inhibition?Potency, incorrect type of agar, concentration.