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Lab Practical I
Microbiology Lab Practical 1
| Question | Answer |
|---|---|
| when do you dis infect your work bench | before and after lab work |
| why do we disinfect the working area before and after work | prevents potential contamination of the scientist, the experiment, and others that follow |
| unnecessary items are at risk of contamination as well. To prevent this, what do we do with them | place unnecessary items in designated areas in the lab |
| ______________ and ________________ are the only items that should be at the work bench | lab manual; lab materials |
| to avoid spills, what should we do with liquid culture tubes | always keep them upright (even when empty), avoid placing them on their side |
| Food and drinks are not allowed in the laboratory primarily because they may ____________________ | transfer microbes into your body |
| while in the lab, what is a really good general rule to follow when it comes to putting things in your mouth | do not put things in your mouth while in the lab |
| What should you do if you need to leave the lab temporarily in the middle of your work | remove lab coat and gloves, wash your hands |
| why would your remove your lab coat and gloves before temporarily leaving the lab | we are assuming that they are potentially contaminated and by wearing them out of the lab is allowing the microbes to leave the laboratory |
| why do we disinfect the workstation prior to working when it is lab protocol that the space is decontaminated after the previous days work | never assume that the person who used the space before your actually decontaminated the space. go a head and disinfect the space just to be sure |
| While removing a clean glass culture tube from its box, you accidently drop the tube, it shatters on the floor. What are the next steps that you should take | sweep up the broken glass and dispose of it in the designated container for broken glass |
| You knock over a culture tube containing bacteria, which shatters on the floor. What should your next steps be? | soak the spill in disinfectant for at least 20 minutes, then dispose of broken glass in a designated container for broken glass |
| when disregarding a contaminated broken glass, where should the remnants be thrown after being disinfected | throw them in the broken glass recepitcal |
| While working with a liquid bacterial culture, you get some on your gloves. What should you do next? | remove your gloves, place them in an autoclave bag, put on a new pair |
| what should you do with the plat of a bacteria that is not being used anymore | place it in the appropriate receptacle for autoclaving |
| all contaminated materials must be ________________ | autoclaved |
| how does the autoclave kill bacteria | with pressurized, high-temp steam |
| what is the definition of selective media | selective media incorporates chemical substances that inhibit the growth of one type of bacteria while permitting the growth of another |
| what is the definition of differential media | differential media incorporates chemical compounds that produce characteristic changes in the appearance of the bacterial colonies |
| what is the definition of enriched media | enriched media is supplemented with highly nutritious materials for the purpose of cultivating fastidious organisms |
| what is phenylethyl alcohol agar selective for | inhibiting the growth of gram-negative organisms and isolates gram-positive organisms |
| what is crystal violet agar selective for | inhibits the growth of gram-positive organisms, used to isolate gram-negative organisms |
| what is NaCl (sodium chloride) agar selective for | inhibits the growth of non-halophilic organisms |
| what type of colonies is NaCl agar used to isolate | Staphylococcus |
| what is the purpose of adding phenol read to a mannitol salt agar | the phenol read permits the differentiation of bacteria based on their ability to produce mannitol |
| what is the purpose of adding the eosin and methylene blue to eosin-methylene blue agar | Eosine and methylene blue are dyes that precipitate on the surface of acid-producing colonies of enteric lactose fermenters creating a metallic green sheen |
| for safety reasons, what should be done with open cuts and wounds on your person | bandaged/covered prior to entering into the lab |
| upon entering lab, unnecessary items go _______________ to lower the risk of contamination | specified locations |
| the only things on the lab bench/work space should be | lab manual and lab supplies are the only things allowed at the work station |
| what should be done about the following items: - food/drink -chewing gum -cosmetics -music players -cellphones | -food/drink (left outside the lab) -chewing gum (spit out) - cosmetics (left with the "unnecessary items") - music players (left with the "unnecessary items") - cell phones (left with the "unnecessary items") |
| to protect the body, what should be worn by the technician | closed-toed shoes, lab coat, safety glasses, and disposable gloves |
| what should technicians with long hair do prior to entering the lab | pull their hair back |
| at what times should technicians wash their hands in the lab | before starting, after completing, before leaving lab temporarily, before putting on a new set of gloves. |
| hands should be washed for ___________________ (length of time) which is long enough to sing __________________ twice | 20 seconds; The "Happy Birthday" song |
| gloves should *always* be worn in the lab if | skin is broken or if the technician has a rash |
| when should gloves be replaced | if the gloves break or if the gloves are visible contamination |
| what types of antimicrobial solutions are used to disinfect the work surface | vesphene or cavicide |
| when should work space be disinfected | before starting or after ending |
| what are the four emergency item locations that need to be learned | first aide kit, eye wash station, safety shower, and fire extiguisher |
| if hazardous liquids are spilt on your person, what needs to be done | hazardous liquids needs to be washed off immediately |
| in case of fire, what is the first step | turn off all gas |
| at the end of lab, what should be done with uncontaminated items | thrown in the trash |
| at the end of lab, what should be done with contaminated tube, plates, and pipets | they need to be autoclaved |
| what type of media categories applies to blood agar (selective/defined/complex/differential/enriched) | Blood agar is DIFFERENTIAL and ENRICHED |
| how is blood agar differential | blood agar distinguishes between hemolytic patterns |
| how is blood agar enriched | blood agar supports growth of many fastidious bacteria |
| Mannitol Salt Agar (MSA) is both selective and differential. It selects for bacteria that are ______________ and differentiates organisms that ferment ________ | halotolerant; mannitol |
| the selective media in MSA is | 7.5% NaCl (sodium chloride) |
| the differential properties of MSA is due to the carbohydrate mannitol and the pH indicator _______________ | phenol red |
| Mannitol fermentation produces acid end-products that __________________ the pH of the surrounding medium | pH decreases |
| the acidic end products of Mannitol fermentation interacts with the pH indicator phenol read, produces ________________ color | yellow |
| an unknown bacterium has grown on an MSA plate and turned it yellow. What can be inferred about the organism | the unknown bacterium is halotolerant and ferments mannitol |
| an unknown bacterium grows on an MSA plate. the plate does not change color. what can be inferred about the bacterium | the unknown organisms are halotolerant and do not ferment mannitol |
| an unknown bacterium does not grow on an MSA plate. what can be inferred about the bacterium | the bacterium is not halotolerant, it is unknown if the organism can ferment mannitol |
| MacConkey is both selective and differential. It selects for bacteria that are _________________ and differentiates organisms that ferment _________________ | Gram-negative; lactose |
| The selective ingredients in MacConkey are _________ and ______________ | crystal violet; bile salts |
| MacConkey's agar differential are due to the carbohydrate __________ and the pH indicator _______________ | lactose; natural red |
| An unknown bacterium grows on a MacConkey plate and turns the plate yellow. what can be inferred about the microorganism | the bacterium is gram-negative and does not ferment lactose |
| An unknown bacterium grows on a MacConkey plate and turns the plate pinkish red. what can be inferred about the microorganism | the bacterium is gram-negative and ferments lactose |
| An unknown bacterium does not grow on a MacConkey plate. what can be inferred about the microorganism | the bacterium is not gram-negative and is likely gram-positive |
| EMB agar is both selective and differential. It selects for bacteria that are _____________ and differentiates organisms that ferment _________________ | EMB agar selects gram-negative and differentiates those that ferment lactose |
| the selective ingredients in EMB agar the dyes are _____________ and ____________ | methylene blue; eosin |
| An unknown bacterium grows on a EMB plate and turns black with a little bit of a sheen. what can be inferred about the microorganism | the bacterium is gram-negative and ferments lactose to produce weak acids |
| An unknown bacterium grows on a EMB plate and turns a little pink. what can be inferred about the microorganism | the bacterium is gram-negative and does not ferment lactose |
| An unknown bacterium grows on a EMB plate and turns metallic sheen. what can be inferred about the microorganism | the bacterium is gram-negative and produces a strong acid |
| C-CNA agar is both selective and differential. It selects for bacteria that are __________________ and differentiates organisms based on their ______________ | gram-positive; hemolytic patterns |
| the selective ingredients in C-CNA agar are two antibiotics: | nalidixic acid and colistin |
| An unknown bacterium grows on a C-CNA plate and turns yellow with dark colonies. what can be inferred about the microorganism | the bacterium is gram-positive and demonstrates complete hemolysis |
| An unknown bacterium grows on a C-CNA plate and there is no color change. what can be inferred about the microorganism | the bacterium on the plate is gram-positive and is incapable of hemolysis |
| An unknown bacterium does not grow on a C-CNA plate. what can be inferred about the microorganism | the bacterium is not gram-positive and is likely gram-negative |
| Plates containing blood, such as blood agar and C-CNA agar, are typically incubated with supplemental carbon dioxide to encourage growth on the plates. What vocabulary term describes organisms that grow best in the presence of carbon dioxide? | capnophiles |
| __________________ are microorganisms that are capable of growing at 4 degrees Celcius | Psychotolerant |
| mesophiles can grow between ________ degrees Celsius and ______________ degrees Celsius | 25 and 37 |
| a _____________ can grow at 55 degrees celsius | thermophile |
| Name the chemical in the fluid thioglycollate broth that acts as a redox potential indicator, turning pink in the presence of oxygen. | resazurin |
| to activate the GasPak jar, water is added to a gas generating envelope that contains: | sodium bicarbonate and sodium borohydride |
| in the GasPak jar, the activated envelope produces two gasses. what are they? | carbon dioxide and hydrogen |
| (in GasPak jars) using the element _____________ as a catalyst, _________ gas combines with free oxygen in the chamber, producing water | pallidium; hydrogen |
| this is a chemical that inhibits the growth of one organism; but allows the growth of another | selective media |
| this media contains nutrients that allow the technician to see how different bacterium metabolize different nutrients | differential media |
| what are the steps in inoculating unknown bacterium on different plates | 1) divide nutrient agar plates into four quadrants and label the bottom, 2) repeat on MSA and EMB plates, 3) streak each culture on the is agar quadrant, 4) incubate the plates inverted at 35 degrees for 24-48 hours |
| what are the importance of different pigments on the nutrient agar plate | helps to identify bacteria |
| what ingredients does the Mannitol Salt Agar (MSA) plate contain | mannitol, 7.5% NaCl, and phenol red |
| MSA plates are selective for | Halotolerant (high salt concentrations) |
| phenol red is a pH indicator that differentiates | mannitol fermentation, produces acid (lowers pH) and phenol red turns yellow |
| EMB stands for | Eosin Methylene Blue |
| the EMB agar contains | peptone, sucrose, lactose, eosin, and methylene blue |
| the dyes in an EMB plate are | eosin and methylene blue |
| the EMB plate is selective for | EMB selects for gram-negative |
| what is the differentiating ingredient in an EMB plate | the sugars (lactose and sucrose) |
| EMB plates differentiates between | the strong fermenters, weak fermenters, and the nonfermenters |
| on the EMB plate, what happens to the fermenters | they change colors. strong fermenters are metallic green, weak fermenters turn dark with a greenish sheen on the edges |
| Bacteria reproduce via | binary fission |
| Generation time is | the length of time it takes for a cell to complete binary fission and form two cells |
| _________________ can be defined as the length of time required to a population to double | Generation time |
| a visual representation of bacterial growth over time | bacterial growth curve |
| name the four distinct growth phases of a bacterial growth curve | lag phase, log phase, stationary phase, and death phase |
| during the stationary phase leading to the death phase | bacterial growth cannot be sustained, nutrients become depleted, waste products accumulate, and future growth |
| open culture systems are known as __________________ | chemostats |
| chemostats can sustain bacterial growth indefinitely as long as | nutrients are provided and wastes are removed |
| there is no growth seen in the lag phase because | this is the phase where the new inoculum is undergoing an intense activity preparing for population growth but no increase in population |
| on a bacterial growth chart, the log phase is | a logarithmic, or exponential increase in population |
| logarithmic growth in log phase is due to | the reproduction by binary fission (bacteria) or mitosis (yeast) |
| on a bacterial growth chart, the stationary phase is | the period of equilibrium; where microbial death and new cell production balance each other out |
| the death phase on a bacterial growth chart is | when the population is decreasing at a logarithmic rate |
| When bacteria are inoculated into a new sterile nutrient broth, their numbers don’t begin to increase immediately. Instead, there is a lag phase that may last for an hour or even several days. Why don’t bacterial numbers increase immediately? | The bacteria must adjust to the nutrient content in the new medium, synthesizing necessary amino acids, growth factors, and enzymes. |
| After a period of rapid growth (log phase), bacterial growth rates will slow and enter the stationary phase. What is happening during stationary phase. | death and growth rates cancel each other out, there are harmful waste products that may be accumulating, cells are likely running out of nutrients |
| if bacteria is being cultured for industrial purposes, they are generally most productive during the _______________ phase | log |
| A chemostat is continuous culture system that is designed to promote and prolong exponential growth and prevent bacteria from entering stationary phase. How might this work? | Chemostats provide a continued source of fresh nutrients and remove wastes and dead bacterial cells. |
| The population size is 1 million cells. The generation time is 30 minutes. Assuming the continuation of log growth, how many cells would there be at 2 hours of growth in log phase? | 16 million |
| under ideal conditions the species with exhibit the (shortest/longest) generation time and grow the (slowest/fastest) | Shortest, Fastest |
| a viable count (or plate count) is | a way of counting bacteria that is living |
| a direct microscopic count | counts all cells whether they are living or dead |
| how are microbial numbers indirectly estimated | turbidity |
| what instrument is used to estimate the turbidity of a liquid bacterial culture | spectrophotometer |
| Consider various methods of counting or estimating the size of bacterial populations. Which of the methods would be most appropriate for gathering data to plot a bacterial growth curve throughout the four phases? | a plate count |
| why is a plate count the most appropriate for gathering data to plot bacterial growth curve | plate counts will provide the number of living cells |
| transferring a inoculum to an agar slant would be done | with an inoculation loop and making a zig-zag streak |
| transferring an inoculum to an agar deep | with an inoculating needle and stabbing the agar deep |
| transferring an inoculum to a broth | using an inoculation loop and swishing the broth |
| how do you perform an isolation streak | streak the first area, starilize, streak second area, starilize, streak third area, then incubate |
| To make 10-fold serial dilutions of the lake water sample, how many mL of diluent should you pipette into tubes A through E? (drawing a picture should help) | 9 mL |
| How many mL of the dilution in Tube C should be transferred into Tube D to create a 10-fold dilution? (draw a picture of A-E tubes) | 1 mL |
| What is the dilution of Tube D? (draw a picture of A - E dilution tubes) | 1:10,000 OR (1/10^4) |
| What is the final dilution on Plate H? (draw a picture of A -H dilution tubes) | 1/10^6 |
| Micropiettes are typically used to pipette small volumes of liquid, and measure volumes in units of microliters (µL). If you were using a micropipette to transfer 0.1 mL of a dilution to a plate, how many microliters would that be? | 100 microLitres |
| what is TNTC | Too Numerous to Count |
| more than _________________ amount of colonies on an agar plate would be considered 'TNTC' | 300 |
| if there are less than ______________ colonies on a plate the results are recorded as 'TFTC' | 30 |
| TFTC stands for | Too few to count |
| 116 colonies on a 10^-5 dilution...calculate the bacterial load in the water sampled | 1.16*10^7 |
| if you were to draw 0.5 microLiters you would use a ____________ pipette | P2 |
| if you were to draw 7.4 microLiters you would use a ____________ pipette | P20 (for 7.4 microLiters) |
| if you were to draw 15.7 microLiters you would use a ____________ pipette | P20 (for 15.7 microLiters) |
| if you were to draw 23 microLiters you would use a ____________ pipette | P200 (for 23 microLiters) |
| if you were to draw 112 microLiters you would use a ____________ pipette | P200 (for 112 microLiters) |
| if you were to draw 621 microLiters you would use a ____________ pipette | P1000 (for 621 microLiters) |
| if you were to draw 201 microLiters you would use a ____________ pipette | P1000 (for 201 microLiters) |
| what are the four sizes of pipettes | P2, P20,P200, P1000 |
| red number on the P20 pipette stands for | the digit after the decimal place in microLiters |
| red number on the P1000 pipette stands for | the digit in the thousands place |
| A scientist is using an objective lens with 40X magnification on his microscope. If the ocular lens magnifies 10X, what is the total magnification being used to visualize the specimen? | 400 X |
| Why is a specimen smaller than 200 nm not visible with a light microscope? | Anything smaller than 200 nm cannot interact with visible light. |
| What happens to the light rays when they hit the specimen? | They are reflected, refracted, or absorbed by the specimen. |
| What is the role of the ocular lens? | To recreate the image in the viewer’s eye |
| What is meant by light rays being divergent? | It is spreading out |
| In a typical brightfield microscope (seen in the animation), at which point does magnification begin? | The objective lens |
| What term best describes the ability to distinguish two separate points that are close together when observing objects through a microscope? | resolution |
| Which style of microscope, used in virtually every school science lab, uses a white light source below the specimen to illuminate the specimen? | Bright-field microscope |
| how do you remove immersion oil from an objective | generously moisten the lens paper with lens cleaner, then wipe around the cone and lens of the objective |
| With respect to the image of a microscope specimen, refraction of light by the objective lens enhances __________. | magnification |
| magnification refers to the | apparent size of the specimen when viewed through the microscope |
| light bends at the ____________ surfaces of the objective lens causing the light rays to diverge and radiate outward | convex surface |
| the _________________ of light creates an image of the specimen that is larger than would normally be seen by the naked eye | divergence |
| In a compound light microscope, what is the function of the condenser? | To concentrate light on the specimen. |
| how does the condenser concentrate the light on the specimen | it gathers the diverging light and concentrates it on the specimen |
| Immersion oil should be cleaned off an objective with which product? | lens paper |
| why should lens paper be used to wipe the lens | lens paper does not scratch the slide or lens, also it is lint free because it has no fibers |
| what is the correct grasp of the microscope arm | both hands, one hand on the arm and one hand on the base |
| You should begin viewing a specimen with what objective lens? | 4X |
| as a general rule, you should start at the (lowest/highest)-power objective to begin viewing under a microscope | start at the LOWEST-power |
| what is one of the main advantages to starting at a lower power | there is a larger field of view, it makes it easy to quickly survey the contents of the slide |
| You have just rotated the 40X objective into position after viewing the specimen with the 10X objective. What would your next step most likely be? | Adjust the fine focus knob. |
| If you view a specimen at 40X total magnification with a 10X ocular lens, then you are viewing the specimen with what objective? | when you are viewing the specimen at 40X then the objective is at 4X |
| name the reagent that serves as the primary stain in the gram-staining procedure | crystal violet |
| what color is crystal violet | purple color |
| name the reagent that serves as the mordant in the gram-staining procedure | iodine |
| what is the function of the iodine (mordant) in the gram staining procedure | helps to fix the crystal violet |
| what is the reagent that serves as the decolorizer in gram staining | 95% ethanol |
| what is the function of 95% ethanol in gram staining | removes the crystal violet-iodine complex from gram negative cells |
| name the reagent that serves as the counterstain in the gram staining procedure | safranin |
| what color is safranin | red/pink |
| Bacillus megaterium is a gram-_______________ rod | Bacillus megaterium is gram-POSITIVE rod |
| staphylococcus xylosus is gram-_____________________ coccus | stapyhlococcus xylosus is gram-POSITIVE coccus |
| escherichia coli is a small gram-__________________ rod | E. coli small gram-NEGATIVE rod |
| name the reagent that serves as the primary stain for the capsule staining procedure | crystal violet primary stain in capsule staining process |
| in this staining procedure, stains the capsule and everything else, including the background purple | the capsule staining proceudre |
| name the reagent that serves as the decolorizer and counterstain in the capsule staining procedure | 20% copper sulfate |
| 20% copper sulfate removes the crystal violet from the ______________ and then counterstains the _______________ a pale blue or clear color | bacterial capsule; capsule |
| what is the procedure for the acid-fast staining procedure | Carbol Fuchsin + steam (primary stains), acid alcohol (decolorizer, and methylene blue (counterstain) |
| what is the procedure for endospore staining | malachite green + steam (primary stain), distilled water (decolorizer) and safranin (counterstain) |
| this biosafety level organisms are well-defined, do not cause harm to humans | Biosafety Level 1 |
| for Biosafety level 1 (BSL 1) what type of safety precautions need to be taken | general lab safety, no extra precautions |
| E. coli, Staphylococcus xylosus, and Bacillus megaterium all belong at what biosafety level | E. coli, Staphylococcus xylosus, and Bacillus megaterium all belong at biosafety level1 |
| this biosafety level has organisms that are associated with moderate human diseases, have a proven treatment, most pathogens are opportunistic | Biosafety Level 2 |
| for biosafety level 2, what precautions need to be taken | some PPE (gloves, lab coats), limiting lab access to certain individuals |
| according the the American Society for Microbiology , environmental samples from water and soil need to be treated at what biosafety level | environmental samples from water and soil need to be treated at biosafety level 2 |
| give some examples of biosafety level 2 organisms | Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella |
| this biosafety level has organisms that are high-risk and have a true potential to infect via aerosols, resulting diseases can be serious or lethal | Biosafety Level 3 |
| define "opportunistic" | organisms that do not normally cause disease in healthy adult humans. But can in children and immunocompromised adults |
| what safety precautions should be taken for biosafety level 3 | double gloves, respirators, disposable surgical scrubs/gowns, work must be done in a safety cabinet and negative pressure environment |
| Mycobacterium tuberculosis and Bacillus anthracis all belong in what biosafety level | Mycobacterium tuberculosis and Bacillus anthracis all belong in biosafety level 3 |
| define the kind of organisms that would be in biosafety level 4 | easily transmitted, very-high risk that are life threatening and have no vaccine or therapy |
| Lab technicians work in impermeable positive pressure "space suits" with an external oxygen supply, as a precaution chemical showers must be taken before exiting the labe | Biosafety level 4 |
| Ebola virus, Marburg Virus, lassa fever virus all be long in biosafety level | 4 |
| what are the "ESKAPE" pathogens | majority of the antibiotic-resistant healthcare-associated infections |
| list the six "ESKAPE" pathogens | Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aerginosa and several species of Enterobacter |
| due to Covid-19 list the additional lab requirements | wear face mask, practice social distancing, no sharing, disinfect eye pieces on microscopes |
| the lab at school only works with what biosafety level(s) | biosafety levels 1 and 2 |
| how does the micropipette work | the internal piston creates a partial vacuum by displacing air, when plunger is depressed |
| when filling the the micropipette plunger, where do you stop? and when letting all the fluid out where do you stop? | when filling, stop at the first stop. when letting out, stop at the second |
| name the two lenses in a bright-field compound microscope | ocular lens and objective lens |
| the ______________ lens is located in the eye piece and the _____________ lens is located in the nose piece | ocular lens (eye piece), objective lens (nose piece) |
| the source of the light in a microscope comes from the | illuminator |
| __________________________ is a two lens system that collects and concentrates light from the illuminator and directs it to the iris diaphram | abbe condenser |
| this item on a microscope regulates the amount of light entering the lens system | iris diaphram |
| what is a mechanical stage | the platform portion of the microscope that has a hole in the center and allows light to pass through it |
| often the mechanical stage contains _____________________ to hold the slide in place | stage clips |
| the objective lens magnifies the _______________ and produces a ______________ that is then magnified by the ocular lens | specimen; real image |
| what is resolving power | the ability of a lens to show two adjacent objects as discrete entities |
| the __________ has the shorter wavelength and is (therefore) better for resolution | blue filter |
| refractive index is | the bending power of light |
| addition of immersion oil does what | diminishes the loss of refracted light and improves resolution |
| what is contrast | the ability to distinguish an object an object from its background |
| what is depth-of-focus | the "thickness" of the sample that appears in focus at a particular magnification |
| as magnification increases depth-of-focus (increases/decreases) | decreases |
| what is par focal | if one objective lens has the object in focus, then you go to the next lens. only minor adjustments are needed to bring the image back to focus |
| as magnification increases, what happens to field of view | field of view decreases with increased magnification |
| what is par central | the ability microscopes have to keep an object in the middle of your field of view when changing from one objective to another |
| what is working distance | the distance between the objective and the slide in a microsope |
| name the three shapes of microorganism cells | coccus (sphere), bacillus (rod), and spiral |
| spiral shaped are divided into two groups based on | flexibility/rigidity |
| spirillum are (stiff/flexible) and spirochete are (stiff/flexible) | spirillum STIFF and spirochete FLEXIBLE |
| coccobacillus are | elongated spheres |
| vibrio are | crescent moon shaped |
| pairs of bacillus are called | diplobacillus |
| chains of bacillus are called | streptobacillus |
| two coccus are called | diplococcus |
| chains of coccus are called | stereptococcus |
| when is a sarcinae produced | when coccus have two planes of cell division and daughters cells are arranged in cubes |
| coccus with multiple planes of cell divisionwill reproduce to form daughter cells arranged in clusters...this is called | staphylococcus |
| what is the purpose of staining | better visualization and classification |
| what is a simple stain | purely to provide contrast, bacterium uptakes stain, background is clear |
| what is a negative stain | the background is stained and the bacterium is not |
| when are negative stains used | observing live bacteria |
| differential stain | allows you to distinguish between bacterial types |
| name the two types of stains used in differential stains | primary stains and counter stains |
| basic stains have a __________________ charge | positive (cationic) |
| acidic stains have a ___________________ charge | negative (anionic) |
| basic stains have an affinity for | negative components of cells |
| name some basic stains | methylene blue, crystal violet, and carbol fuchin |
| acidic stains have an affinity for | positive components of cells |
| name some acid stains | nigrosin, India ink, and picric acid |
| cell walls are ______________ charged | cell walls are negatively charged |
| what type of dyes are attracted to cell walls | basic dyes (like methylene blue, crystal violet, and carbol fuchsin) are attracted to cell walls, while acidic dyes are repulsed by cell walls |
| what is a bacterial smear | when bacteria is placed on a glass slide and prepared with a specific staining process |
| what is the point of heat fixation | kills the organism, causes organism to adhere to the plate, and alters organism to more readily accept dyes |
| if the slide is too wet before heat fixation | organisms are boiled and destroyed |
| if slide is not heat fixed enough | the organisms will wash off during staining process |
| if the heat is fixed too much | organisms may become damaged and cells won't stain correctly |
| after rubbing a culture on to a slide, what do you do | sterilize the loop completely and let the smear air dry completely |
| an alternative to aid drying a bacterial smear is | using a slide warmer on LOW HEAT |
| what is the incinerator method for heat fixing a slide | holding the slide with a wooden clothes pin of the barrel of a hot incinerator |
| what is the Bunsen burner method | hold the slide with a wooden clothes pin, and a pass through the flame |
| a gram-stain is a _______________ stain that distinguishes bacteria based on cell wall properties | gram-stains are differential stains |
| bacterial walls are composed of primarily | peptidoglycan |
| what are the two main groups that bacteria can be differentiated into based on the gram-stain results | whether or not peptidoglycan is present in their walls |
| organisms that have a thick layer of peptidoglycan will be Gram-________________ | thick layer of peptidoglycan will be Gram-POSITIVE |
| organisms with thin layer of peptidoglycan are Gram-_______________ | thin layer of peptidoglycan are Gram-NEGATIVE |
| gram negative organisms have | thing layer of peptidoglycan plus an additional outer membrane |
| what are the steps in gram-staining | cover the smear in crystal violet, rinse with water, cover with iodine, rinse with water, rinse with the decolorizer (95% ethanol), cover smear with safranin, rinse |
| capsule stain is a type of _________________________ stain which selectively stains bacterial capsules | capsule stains are differential |
| what is a capsule | a substance that is synthesized in the cytoplasm and secreted outside the cell where it surrounds the bacterium |
| in Streptococcus pneumoniae and Nisseria meningitidis capsules are associated with _________________ | virluence |
| for some virluent bacteria, capsules are important for | providing a mechanism for these pathogens to evade the host immune system |
| what do heat and water do to capsules during laboratory procedures | heat and water dislodges capsules |
| capsule stains (do/do not) contain a mordant | capsule stains DO NOT contain a mordant |
| in capsule stains, 20% copper sulfate serves two purposes | decolorizing stain and the counter stain |
| describe the process of capsule staining | cover the smear with crystal violet, rinse with 20 % copper sulfate, let air dry, DO NOT USE WATER |
| acid-fast stain is a differential stain that distinguishes bacteria based on | cell wall properties |
| bacteria which are acid-fast are also gram-____________________ | acid-fast bacteria are also gram-POSITIVE |
| acid fast bacteria have a waxy substance called ______________________ in their walls, which compromises up to ________% of the cell wall components | mycolic acid; 60% |
| acid-fast bacteria also contain polymers called __________________ and _________________________ in their cell walls | arabinoglactan and lipoarabinomannan |
| two species that are acid-fast include | Mycobacterium tuberculosis and Mycobacterium leprae |
| what is the purpose of phenol in acid-fast staining | phenol solubilizes the cell wall and allows the stain to enter the cell |
| in order for the carbol fuchsin and phenol to penetrate the waxy cell wall _______________ is applied | heat |
| the decolorizer in fast acid cells is _________________, which decolorizes all cells except _____________ | acid alcohol; acid-fast cells |
| counterstain for fast acid is | methylene blue |
| what is the procedure for fast acid stain | apply carbol fuchsin to soak a paper towel, put slid over hot plate, rinse with distilled water, rinse with acid alcohol, rinse with distilled water, cover slide in methylene blue |
| the endospore stain is differential for | bacterial endospores or spors |
| what are the two generas that have endospores | clostridium and bacillus |
| what are spores | metabolically inactive cells |
| why are spore important | highly resistant to hostile chemical and physical conditions |
| the process that of producing spores | sporulation |
| when does sporulation occur | when the environmental conditions become unfavorable for normal vegetative growth |
| the tough outer structure of the spore is composed of ________________ | keratin |
| how long will bacteria remain suspended as an endospore | spores can remain in this state until conditions are more favorable |
| what does the term endospore come from | the spore structure that is contained within a mother cell |
| what procedures do you follow when performing an acid-fast stain | apply malachinte green to a paper towel and steam, rinse with distilled water, cover the safranin, rinse with water. |
| what is subculturing | the transferring of microorganisms from one media type to another |
| what is an agar slant | solid media in a test tube with a slanted surface |
| what is an agar deep | a solid media in a test tube where the surface does not have a slant |
| how are agar deeps typically inoculated | stabbing the media with a sterile needle |
| agar plate is | solid media, in a petri dish, provides optimal surface for cultur e growth |
| how are agar slants and agar plates inoculated | with an inoculated loop streaking the surface |
| what instruments can be used to inoculate a broth cultur | sterile loop, needle, or pipette |
| steps to gathering an inoculum from a tube | starilize the loop/needle, open tube, pass through flame, gather inoculum, pass through the flame, close the tube |
| what is the purpose of isolating of pure cultures | to separate mixed cultures of multiple different bacteria and form single, isolated colonies of each bacterial species |
| cultures that are visible on the surface of solid media are called | colonies |
| a colony can form on a plate when | a single viable microbe is inoculated onto the surface of the plate and reproduces until there are enough cells to form a visible colony |
| a colony should (in theory) represent a pure culture since... | a colony is the theoretical form of a single cell |
| what are the two most common methods to obtain a single, isolated colony on a petri dish | Four Way streak for isolation and a T streak method for isolation |
| in both the Four way streak method and the T streak method, why is it easier to obtain an individual colony | because the sample is diluted each time you streak the surface of the diash |
| steps for quadrant streak method | make four quadrants and label (1-4), gather the inoculum and spread onto quad. 1, sterilize loop, spread over quad. 2. passing through 1, a couple times. repeat this step for quad. 3 and 4 |
| steps for T-streak method | divide plate into "T," streak the inoculum on the top, sterilize the loop, streak sector 2 passing through sector 1, repeat with sector 3 |
| what is one of the best ways to quantify microorganisms | viable plate count |
| what is the major step necessary to do a viable plate count | serial dilutions |
| what are serial dilutions | a step-process that sequentially dilutes bacterial cultures |
| what is the total dilution plated for a 0.1 mL of a 10^-6 dilution that was plated | 10^-7 |
| what are the three cardinal temperatures for microorganism growth | minimum growth temp., optimum growth temp, maximum growth tem |
| what is the minimum growth temp. | the lowest temperature at which a specific organism will grow and reproduce |
| what is the optimal growth temperature | the temperature at which a specific organism exhibits maximum growth and reproduction |
| what is the maximum growth temperature | the highest temp. at which a specific organism will grow and reproduce |
| psychrophile can grow between what temperatures | -5 to 20 decgrees Celcius |
| a mesophile can grow at what temperature range | 20 to 45 degrees Celcius |
| what are psychrotolerante or psychrotroph organisms | mesophiles that can grow at temperatures below 10 degrees Celcius |
| what is the temperature range for thermophiles | 45 to 80 degrees Celcius |
| some archaea are classified as hyperthermophiles and can go at what temperature | over 80 degrees Celcius |
| what are the major classes of bacterial oxygen requirements | aerobes, microaerophiles, obligate anaerobes, aerotolerant, facultative anaerobes |
| obligate aerobes | require atmospheric oxygen (20%) and use oxygen as the final electron acceptor in the electron transport chain |
| microaerophiles | require atmospheric oxygen at below atmospheric pressure (roughly 2-10%) |
| why can't microaerophiles live at regular atmospheric pressure | while they do use oxygen as the final electron acceptor in the electron transport chain, they have limited ability to neutralize oxygen |
| obligate anaerobes | cannot survive in the presence of oxygen |
| why can't obligate anaerobes live at regular atmospheric oxygen levels | they lack the enzymes necessary to break down the toxic by-products of Oxygen |
| in obligate anaerobes, the final electron acceptor is | something other than oxygen |
| aerotolerant anaerobes | grow equally well in the presence of the absence of oxygen |
| what kind of enzymes do aerotolerant anaerobes | they possess the enzymes necessary to neutralize toxic oxygen by products but they never se oxygen as a final electron acceptor |
| facultative anaerobes | live in either presence or absence of oxygen, but they prefer oxygen so they can carry out aerobic respiration with oxygen as a final electron acceptor to maximize ATP yields |
| if oxygen is not available, what do facultative anaerobes do | the organism will use other elements as electron acceptors (nitrate or fumarate) |
| specialized methods are necessary to culture (aerobically/anaerobically) | specialized methods are necessary to culture organisms ANAEROBICALLY |
| what is the fluid thioglycollate brothe | a method growing cultures anaerobically, which is a reducing medium |
| fluid thioglycollate broth contains _______________________________________ , which reacts with molecular oxygen keeping free oxygen at low levels | sodium thioglycollate |
| the sodium thioglycollate creates a ______________________ in the tube | redox potential |
| what is the redox potential | with higher levels of oxygen at the top of the tube and the complete absence of oxygen at the bottom of the tube |
| what is resazurin | the redox potential indicator in fluid thioglycollate broth, this turns pink in the presence of oxygen |
| what is the use of GasPak Jar | a method for growing cultures anaerobically |
| how are anaerobic conditions are created in a GasPak Jar | by adding the water to a gas generator envelope containing sodium borohydride and sodium bicarbonate prior to sealing the chamber |
| water reacts with the ________________ and __________________ in the GasPak Jar creating __________ gas and ______________ | sodium borohydride and sodium bicarbonate, hydrogen and carbon dioxide |
| in the GasPak Jar Hydrogen gas is produced from the | sodium borohydride |
| in the GasPak Jar carbon dioxide gas is produced from the | sodium bicarbonate |
| how does the GasPak jar create an anaerobic environment | the hydrogen gas binds with water and turns all free oxygen into water |
| _________________ is the catalyst in the GasPak Jar for creating water and an anoxic environment | palladium |
| what is a simpler and more modern method for creating an anaerobic environment | AnaeroPack box |
| describe the AnaeroPack | an anaerobic environment is generated after inoculated media are sealed in chamber , the envelopes used do not require a water or a catalyst. |
| the main ingredient in the envelope of the AnaeroPack is | Asorbic Acid (Vitamin C) |
| what happens to the contents of the envelope in the anaeropack when exposed to air | the chemicals become activated, causes the production of CO2 and the rapid absorption of molecular O2 |
| what types of media are used to help identify and classify bacteria | selective media, differential media, and enriched media |
| what are some examples of selective media | Mannitol Sale agar (MSA), MacConkey agar, Eosin-methylene blue (EMB) and columbia C-CNA agar |
| most differential media contains a substrate and a chemical indicator, usually ________________ | pH indicator |
| what are some examples of differential media | MSA, DNAse agar, blood agar, MacConey agar, and eosin-methylene blue agar |
| what are some examples of enriched media | blood agar and chocolate agar |
| a pH indicator is a _________________ chemical that produces different pH values , giving a visual representation of pH of a substance as to whether it is acidic, basic, or neutral | halochromic |
| what colors is bromocresol purple when it is acidic, neutral, or basic | yellow (acidic), purple (neutral), N/A (basic) |
| what colors is methyl red when it is acidic, neutral, or basic | red (acidic), yellow (neutral), N/A (basic) |
| what colors is bromothymol blue when it is acidic, neutral, or basic | yellow (acidic), blue-green (neutral), Prussian Blue (basic) |
| what colors is phenol Red when it is acidic, neutral, or basic | yellow (acidic), red (neutral), Hot Pink (basic) |
| MSA selects for _____________________ and differentiates for organisms that __________________ | halotolerant; fermenters of mannitol |
| fermentation of mannitol in MSA | decreases the pH, causing the differential medium (phenol red) to turn yellow |
| what causes hemolysis in blood agar | some bacteria produce exotoxins (hemolysins) |
| what is the differentiation tool in blood agar | the degree in hemolysis |
| name the three types of hemolysis | beta hemolysis, alpha hemolysis, gamma hemolysis |
| ________ is the complete lysis of red blood cells and hemoglobin; results in complete clearing of the blood around colonies | beta hemolysis |
| ________________ is the partial lysis or red blood cells and hemoglobin. this results in a greenish-grey or greenish-yellow discoloration of the blood around the colonies | alpha hemolysis |
| _______________ is no hemolysis with no change in the medium | gamma hemolysis |
| what chemicals are in the MacConkey agar | crystal violet, bile salts, lactos and natural red |
| MacConkey agar is selective for ___________________ and differentiates for ______________________ | gram-negative organisms, lactose fermenters |
| E. coli on a MacConkey (indreases/decreases) the pH on the media turning it | decreases; pinkish-red |
| on a MacConkey plate, if a colony does not ferment lactose then | the colonies with be colorless or takes on the color of the medium |
| what ingredients does eosin methylene blue contain | eosing, methylene blue, and lactose |
| EMB plates select for _____________ and differentiate ________________ | EMB plates select for gram negative and ferment lactose |
| in an EMB plate, small amount of acid production turn the plate | pink colored colonies |
| in an EMB plate a large amount of an acid production turn the plate | metallic greenish sheen |
| the Columbia C-NA (CCNA) agar selects for _________________ and differentiates between | gram-positive organisms and hemolytic patterns |
| the CCNA media contains two _______________ called colistin and nalidixic acid | antibiotics |
| Biosafety Level 2 precautions include | all general lab safety rules plus requiring the use of PPE such as disposable gloves and lab coats. Lab access is limited to trained individuals |
| Biodafety Level 1 precautions include | general lab safety rules such as no eating or drinking, prohibition of mouth pipetting, practicing aseptic technique and proper disposal of sharps and microbiological waste |
| a _______ is a poly saccharide of polypeptide layer produced by some cells that can aid in attachment, protection from desiccation or increases virulence | Capsule |
| fimbriae are | short, fine, hair-like appendages that aid in attachment |
| pili are | specialized structures often involved in the transfer of genetic material from one bacteria to another |
| the ________________ was a long rotating structures involved in motility | Flagella |
| the ______________________ is responsible for the corkscrew movement of certain types of cells | axial filiment |
| the endospore is a | highly resistant structures that are metabolically inactive and can remain inactive and can remain in the environment a very long time |
| a polymer that is found in cell walls is | peptigoglycan |
| name some cellular components that are found in all cells | ribosomes, cell membrane, cytosol, DNA, and cytoskeleton |
| when using a microscope, when would you use the coarse focus knob | on the lowest objective only |
| you perform a gram stain on a pure culture of bacterial cells that you knew ahead of time should stain purple at the end. However, upon viewing under the microscope you notice that most of the cells appear pink. Name a reason that may have lead to this. | over decolorizing, forgot the primary stain (crystal violet) , cultures are too old |
| name a couple common genera that stains acid-fast | Mycobacterium or Norcardia |
| when viewing an endospore stain under a microscope, what color will be vegetative cell appear | pink |
| During the streak plate method, why is it important to sterilize the inoculating loop between streaking each quarter of the plated | to reduce the number of bacterial on the plate so that isolated colonies form on the final quadrant |
| you have obtained a pure broth culture of suspected E. coli bacteria and spread it on a Salmonella and Shingella agar plate then incubated for 48 hours. how to determine if the broth culture is positive for salmonella | Salmonella colonies appear black on this agar type |
| you are asked to make a 10-fold bacterial dilution in a final volume of 40 mL . Indicate what volume is needed of bacteria and sterile water | Bacteria = 4 mL and Sterile water = 36 mL |
| the time it takes for a bacterial population to double in size | generation time |
| why doe we report the viable count as "colony forming units per m:" rather than "cells per mL" | A colony may have originated from two cells that were very close together when they were introduced to the agar plate. Both of the cells formed the resultant colony. the total number of colonies may be under-reporting the number of cells |
| you have performed an isolation streak to obtain a pur culture. Assuming the isolation streak was performed properly, what is the starting number of cells for this calculation | 1 Cell forming units |
| what is the formula for cell growth | N(t) = N(0) * 2^n |
| if the initial unit for an isolation streak is 1 Cell what is the total number of cells in an isolated colony (if generation time is 30 minutes, and given 5 hours to grow) | 1024 cells |
| name two media types that can be used to determine lactose fermentation | MSA and MacConkey |
| name one indicator that there is a contaminant present in the sample when analyzing a fermentation data set | Contaminates usually grow very rapidly and can lead to: -High biomass relative to product and - high levels of CO2 |
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